Liposome-OBP conjugates for odour reduction and fragance release
Abstract
A atividade diária e o exercício físico são responsáveis pela produção de odores corporais desagradáveis que podem causar ansiedade e embaraço social. A procura de novas soluções que previnam o desenvolvimento desses odores é atualmente objeto de interesse para as indústrias da cosmética e têxtil. Nos mamíferos, as proteínas de ligação a odores (OBPs) são responsáveis pelo transporte de moléculas odoríferas do muco nasal aos recetores olfativos. As OBPs são proteínas extracelulares com uma estrutura robusta e estável em barril β, com grande capacidade para ligar a diferentes ligandos. Estas características têm sido foco de diferentes trabalhos de modo a compreender os mecanismos inerentes à sua função na natureza e a desenvolver aplicações biotecnológicas avançadas. Os resultados levaram-nos a estudar as OBPs como uma solução elegante para prevenir e/ou remover odores desagradáveis dos têxteis, através da captura de odores e libertação controlada de fragrâncias. Inicialmente, a OBP de porco (pOBP) foi fundida com três péptidos de penetração celular (CPPs). Estas proteínas (OBP::CPPs), em conjunto com lipossomas, foram usadas como transportadores e reservatórios num sistema avançado de captura de moléculas odoríferas. A pOBP foi também fundida com o péptido SP-DS3, com e sem o espaçador GQ20 para a ancoragem na membrana lipídica de lipossomas. A transdução/captura de 1-aminoanthraceno (1- AMA, ligando modelo fluorescente) para o interior dos lipossomas revelou ser dependente da proximidade da proteína à membrana lipídica. Estes trabalhos permitiram o desenvolvimento de dispositivos para a encapsulação de fragrâncias e captura de odores pelos lipossomas. Outras proteínas, a OBP truncada com as mutações F44A e F66A, e a OBP::GQ20::SP-DS3, apresentaram uma afinidade ao 1-AMA diferenciada e dependente da temperatura Recentemente, foi desenvolvido um “têxtil inteligente” pela funcionalização do tecido com OBP::GQ20::CBM (OBP fundida com o espaçador GQ20 e um módulo de ligação a carbohidratos). O têxtil funcionalizado revelou capacidade de libertação controlada de fragrâncias em resposta à transpiração (suor).
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Universidade do Minho Escola de Engenharia Filipa Daniela Gomes Gonçalves Liposome-OBP Conjugates for Odour Reduction and Fragrance Release janeiro de 2021 UMinho | 2021 Filipa Daniela Gomes Gonçalves Liposome-OBP Conjugates for Odour Reduction and Fragrance Release
Filipa Daniela Gomes Gonçalves Liposome-OBP Conjugates for Odour Reduction and Fragrance Release Doctoral Thesis Doctoral Degree in Chemical and Biological Engineering Supervisor by: Doutor Artur Jorge Araújo Magalhães Ribeiro Doutora Carla Manuela Pereira Marinho da Silva January 2021
ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ [Esta é a mais restritiva das nossas seis licenças principais, só permitindo que outros façam download dos seus trabalhos e os compartilhem desde que lhe sejam atribuídos a si os devidos créditos, mas sem que possam alterá-los de nenhuma forma ou utilizá-los para fins comerciais.]
iii Agradecimentos Quero agradecer aos meus orientadores todo o apoio, aprendizagem e generosidade durante este percurso. Aprendi muito convosco e levo para futuro boas lembranças e um enorme desenvolvimento pessoal e profissional. Muito obrigada. Não posso deixar também de agradecer aos meus colegas pela ajuda e pelo bom ambiente vivido no laboratório. Agradeço à Universidade do Minho, em particular ao centro de Engenharia Biológica pelo suporte disponibilizado, instalações e regras de aprendizagem envolvidas, dando particular atenção aos técnicos e o seu apoio. Quero ainda agradecer à Fundação para a Ciência e Tecnologia (FCT, SFRH/BD/114684/2016) pelo apoio financeiro ao longo de todo o percurso académico, sem ele não seria possível concluir este objetivo. Por último, gostava de agradecer à minha família todo o apoio e incentivo na concretização desta meta académica.
iv Statement of Integrity I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho.
v Conjugados lipossomas-OBP para a redução de odores e libertação de fragrâncias Resumo A atividade diária e o exercício físico são responsáveis pela produção de odores corporais desagradáveis que podem causar ansiedade e embaraço social. A procura de novas soluções que previnam o desenvolvimento desses odores é atualmente objeto de interesse para as indústrias da cosmética e têxtil. Nos mamíferos, as proteínas de ligação a odores (OBPs) são responsáveis pelo transporte de moléculas odoríferas do muco nasal aos recetores olfativos. As OBPs são proteínas extracelulares com uma estrutura robusta e estável em barril β, com grande capacidade para ligar a diferentes ligandos. Estas características têm sido foco de diferentes trabalhos de modo a compreender os mecanismos inerentes à sua função na natureza e a desenvolver aplicações biotecnológicas avançadas. Os resultados levaram-nos a estudar as OBPs como uma solução elegante para prevenir e/ou remover odores desagradáveis dos têxteis, através da captura de odores e libertação controlada de fragrâncias. Inicialmente, a OBP de porco (pOBP) foi fundida com três péptidos de penetração celular (CPPs). Estas proteínas (OBP::CPPs), em conjunto com lipossomas, foram usadas como transportadores e reservatórios num sistema avançado de captura de moléculas odoríferas. A pOBP foi também fundida com o péptido SP-DS3, com e sem o espaçador GQ20 para a ancoragem na membrana lipídica de lipossomas. A transdução/captura de 1-aminoanthraceno (1AMA, ligando modelo fluorescente) para o interior dos lipossomas revelou ser dependente da proximidade da proteína à membrana lipídica. Estes trabalhos permitiram o desenvolvimento de dispositivos para a encapsulação de fragrâncias e captura de odores pelos lipossomas. Outras proteínas, a OBP truncada com as mutações F44A e F66A, e a OBP::GQ20::SP-DS3, apresentaram uma afinidade ao 1-AMA diferenciada e dependente da temperatura Recentemente, foi desenvolvido um “têxtil inteligente” pela funcionalização do tecido com OBP::GQ20::CBM (OBP fundida com o espaçador GQ20 e um módulo de ligação a carbohidratos). O têxtil funcionalizado revelou capacidade de libertação controlada de fragrâncias em resposta à transpiração (suor). Palavras-chave: libertação de fragrâncias, lipossomas, proteínas de ligação a odorantes, redução de odores, têxtil inteligente
vi Liposome-OBP conjugates for odour reduction and fragrance release Abstract The daily activity and physical exercise are responsible for the generation of unpleasant body odors that may cause social unrest and embarrassment. The search for new solutions to prevent the development of these odors is nowadays a subject with great interest for cosmetic and textiles industries. In mammals, odorant-binding proteins (OBPs) are responsible to transport odorant molecules across the aqueous nasal mucus until the olfactory receptors (ORs). OBPs are small extracellular proteins with a robust and stable three-dimensional structure in β-barrel with great ability to bind differentiated ligand molecules which has driven the research to understand the mechanisms underlying the OBP function in nature and the development of advanced biotechnological applications. These features inspired us to study OBPs as an elegant solution to prevent and/or remove unpleasant odors from textiles, by the entrapment of odors and the controlled release of fragrances. Firstly, porcine OBP (pOBP) was fused with three cell penetrating peptides (CPPs). A new methodology using liposomes as reservoirs and OBP::CPPs as carriers was developed as an advanced system to capture odorant molecules. pOBP was also fused with an anchor peptide (SP-DS3), without and with a spacer GQ20, and the liposomes were produced anchoring these new fusion proteins in the lipid membrane. The transduction of 1-aminoanthracene (1-AMA, a fluorescent ligand model) into the liposomes revealed to be driven by the proximity of the protein to the liposomal membrane. Both works showed the development of an efficient device for the encapsulation of fragrances or capture of unpleasant odors inside of the liposomes. Other two proteins, truncated OBP with mutation F44A and F66A, and OBP::GQ20::SP-DS3 presented differentiated 1-AMA binding behavior depending on the temperature. Further a smart fabric was developed by functionalization with OBP::GQ20::CBM (OBP fused with a spacer GQ20 and a carbohydrate-binding module). The functionalized fabric exhibited controlled release of fragrances triggered by perspiration (sweat). Keywords: fragrance release, liposomes, odorant-binding protein, odor reduction, smart textile
vii Index Agradecimentos ......................................................................................................................... iii Resumo .....................................................................................................................................v Abstract .................................................................................................................................... vi Index ....................................................................................................................................... vii List of figures ........................................................................................................................... xiii List of tables ............................................................................................................................ xxi Chapter 1 Objectives and outline ................................................................................................................ 1 Chapter 2 Mammalian odorant-binding proteins ........................................................................................... 4 1. Introduction ............................................................................................................................ 5 2. Mammalian olfactory transduction system ............................................................................... 5 3. The role of mammalian odorant-binding proteins ..................................................................... 6 4. Physicochemical and structural properties of OBPs ............................................................... 10 5. Binding affinity and selectivity of mammalian OBPs ............................................................... 13 6. Mechanistic insights about OBP mode of action .................................................................... 26 6.1. The nature of the ligand influences the binding site at OBP surface ................................. 26 6.2. Understanding the mammalian OBP/odorant interactions .......................................... 31 7. Applications of mammalian OBPs ...................................................................................... 32 7.1. OBPs as biosensors ....................................................................................................... 33 7.1.1. Biosensors for control of water, air and soil contamination ........................................... 33 7.1.2. Biosensors for control of wine and food quality ............................................................ 34 7.1.3. Biosensors for explosives and drugs detection ............................................................. 35 7.1.4. Biosensors for medical diagnosis ................................................................................ 36 7.2. OBPs as capture and release devices of odorant molecules ............................................ 36
xiv > 0.5 in the Gonnet PAM 250 matrix), a ‘.’ (period) indicates conservation between groups of weakly similar properties (scoring = < 0.5 in the Gonnet PAM 250 matrix). The OBP structures observed in (B) were retrieved from PDB. In the figure are presented the OBPs from: human Homo sapiens OBPIIa (PDB ID: 4RUN), boar Sus scrofa (1GM6); giant panda Ailuropoda melanoleuca (5NGH); rat Rattus norvegicus OBP (3ZQ3); pig OBP isolated from Sus scrofa (1DZK); OBP from bovine Bos taurus (1OBP); and an isoform of OBP-1 from rat Rattus norvegicus (3FIQ). The primate order is represented by red box, the artiodactyla order is represented at light green box, the carnivora order is represented by dark blue box and the rodentia order is represented by light grey box. ................................................... 11 Figure 2.5. Cartoon diagram of porcine OBP protein (pOBP). The figure indicates the localization of Trp16 residue (red, W16), five Tyr residues (blue, Y20, Y52, Y78, Y82 and Y92), and the disulfide bridge between Cys63 and Cys155 (yellow). The figure was retrieved from Staiano et al., 200756, constructed on the basis of pOBP structure given in file 1A3Y.pdb. ................................................ 12 Figure 2.6. Cartoon illustration of pig OBP (PDB ID: 1DZK). The elements of the secondary structure are indicated (β-stands A-H, α-helix and Loops L1-L7). The figure was created using PyMOL. ......... 27 Figure 2.7. Properties and applications of mammalian OBPs. ........................................................ 33 Figure 3.1. Cartoon representation of OBP wt and OBP fused with CPPs, complexed with 1-AMA. The OBP is shown in grey scale, 1-AMA in magenta and the CPPs in rainbow colors; the position of the ligand on each OBP system was settled through a standard molecular docking procedure with AutoDock4 and represents the best binding energy. ...................................................................... 42 Figure 3.2. Experimental procedure to evaluate the 1-AMA transduction into liposomes. ................ 47 Figure 3.3. Different views of OBP wt complexed with 1-AMA. (A) Protein is represented in green and 1-AMA in magenta spheres. (B) Model lipid membrane generated with MemGen. Head groups are represented by spheres (blue for nitrogen, red for oxygen and orange for phosphorus), and aliphatic chains are represented in green. (C) WHAM histogram on states superposition of umbrella sampling simulated windows. ...................................................................................................................... 50 Figure 3.4. (A) SDS-PAGE electrophoresis of 100 μM of OBP wt, OBP::Pep-1, OBP::pVEC, OBP::Tat and Precision Plus ProteinTM molecular weight; (B) Theoretical (by SnapGene® 3.0.3) and quantified (by MALDI-TOF) mass of OBP::CPPs. .................................................................................................. 53
xv Figure 3.5. Circular dichroism spectra of (A) OBP wt and OBP::CPPs; a close-up of pVEC is highlighted; (B) CPP peptides and (C) OBP::CPPs in the presence and absence of 1-AMA; for a better visualization of CD spectrum of CPPs and pVEC, different scales were used. ..................................................... 54 Figure 3.6. Binding curves obtained by measuring the fluorescence of 1 μM OBPs::CPPs in 50 mM Tris–HCl, pH 7.5, at equilibrium with several concentrations of 1-aminoanthracene. The dissociation constants were obtained at 37 °C by mathematical fitting of data.143 Values are the mean ± SD of 3 independent experiments. ............................................................................................................. 55 Figure 3.7. Fluorescence spectra of OBP wt, OBP::Pep-1, OBP::pVEC and OBP::Tat, obtained after 1 h of incubation at 37 °C (Ex. 295 nm). ......................................................................................... 58 Figure 3.8. Free energy profile of 1-AMA in DOPE:DSPE:CHOL membrane and representative snapshots of 1-AMA moving in membrane normal axis, as background; water molecules and sodium ions were omitted for better visualization; 1-AMA molecule is shown in magenta spheres and OBP in green cartoon. For membrane, blue for nitrogen, red for oxygen, orange for phosphor and green for carbon. ......................................................................................................................................... 60 Figure 4.1. (A) Representation of the mammalian olfactory system. The odorant-binding proteins (OBPs) bind and carry the odor to the olfactory receptors (ORs), triggering an intracellular signaling cascade. (B) Representation of an anchorage-based system composed by fusion OBPs and liposomes for the entrapment and transport of molecules, such as 1-aminoanthracene (1-AMA). Figure 4.1A was created based on Firestein (2001)13 and Sankaran et al. (2012).16 .................................................. 65 Figure 4.2. Characterization of engineered OBPs by SDS-PAGE gel electrophoresis (A); MALDI-TOF (B); binding dissociation constants (Kd) (C); CD spectroscopy (D) and I-TASSER structural models (E). In (A) is presented the run of purified wild-type OBP (1), OBP::SP-DS3 (2) and OBP::GQ20::SP-DS3 (3); M, Precision Plus ProteinTM Standards. In (B), theoretical mass including the His-tag and the m/z values are indicated; the wild-type OBP has 20054.64 Da, the OBP::SP-DS3 has 21357.16 Da whereas the OBP::GQ20::SP-DS3 has 25066.71 Da. In (C) the constants were determined after binding of the proteins with different concentrations of 1-AMA, 1 h at 37 °C. In (E) the arrows indicate the alterations added to wild-type protein. ............................................................................................................ 71 Figure 4.3. Relative fluorescence versus 1-AMA concentration for OBP wt, OBP::SP-DS3 and OBP::GQ20::SP-DS3 when incubated at 37 ºC for 1 h (A). 1-AMA fluorescence competitive binding assay. Fluorescence emission spectra were recorded at 37 °C with 2 μM of 1-AMA in the presence of 1 μM
xvi of protein, pH 7.5 for 1 h, followed by addition of increasing concentrations of (R)-(-)-carvone 98% and (S)-(+)-carvone 96%, and incubated for 1 h at 37 °C (B). Excitation and emission wavelengths were 295 and 481 nm, respectively. Values represent the mean ± SD of 3 independent experiments. .... 72 Figure 4.4. Size distribution of liposomes functionalized with OBP::SP-DS3 (A) and with OBP::GQ20::SPDS3 (B) and for nonfunctionalized liposomes (C). (1) and (2) are independent measurements of replicates. .................................................................................................................................... 73 Figure 4.5. SDS-PAGE gel of liposomes functionalized with OBPs: (1) 45 μM OBP::SP-DS3; (2) liposomes with anchored OBP::SP-DS3; (3) nonanchored OBP::SP-DS3; (4) 45 μM OBP::GQ20::SP.DS3; (5) liposomes with anchored OBP::GQ20::SP-DS3; (6) nonanchored OBP::GQ20::SP-DS3 and (M) 4 μL Precision Plus ProteinTM Standards (BioRad). .................................................................................. 74 Figure 4.6. Physicochemical characterization of nonfunctionalized liposomes (A, D) and liposomes containing the proteins OBP::SP-DS3 (B, E) and OBP::GQ20::SP-DS3 (C, F). Size, polydispersity index (PDI) and surface charge (ζ-potential) were measured using a Zetasizer Nano ZS. Values represent the mean ± SD of 3 independent experiments. .................................................................................... 75 Figure 4.7. 1-AMA percentage transduced into liposomes and bound to the OBPs functionalized in liposomes after 1 h of incubation at 37 °C. (A) Experimental procedure to evaluate the amount of 1AMA transduced into the liposomes and bound to protein-functionalized liposomes. After incubation of liposomes with 1-AMA at 37 °C, the free ligand is removed using a gel filtration chromatography column with a 5 kDa cut-off. The amount of 1-AMA in the liposomes was measure by the fluorescence emission at 600 nm. (B) Experimental procedure to evaluate the amount of 1-AMA transduced into the liposomes and bound to wild-type OBP. The liposomes were incubated with protein and 1-AMA at 37 °C for 1 h. Then the free protein, free ligand and 1-AMA/OBP complex were separated from liposomes using a membrane 100 kDa cut-off. The free ligand is removed using a gel filtration chromatography column with a 5 kDa cut-off; 1-AMA in the liposomes was measure by the fluorescence emission at 600 nm. (C) Percentage of 1-AMA in liposomes and binding to proteins determined by fluorescence emission. The values are the mean ± SD of 2 independent experiments. ....................................... 77 Figure 5.1. Sequences alignment of OBP wt, tOBP and OBP::GQ20::SP-DS3. The alignment was performed using the CLUSTAL O (1.2.4) multiple-sequence alignment program. In red are highlighted the alanine residues mutated in tOBP; in blue is indicated the linker composed by glycine and glutamine repetition added to OBP::GQ20::SP-DS3 protein; in green is highlighted the SP-DS3 peptide. 82
xvii Figure 5.2. Opposite temperature-dependent affinities of tOBP and OBP::GQ20::SP-DS3 to 1aminoanthracene (1-AMA). In (A) are presented the different binding affinities of tOBP and OBP::GQ20::SP-DS3. (B) is the schematic presentation of OBP’s competitive temperature-dependent mechanism; tOBP is presented in grey; OBP::GQ20::SP-DS3 is presented in magenta and 1-AMA is presented in green. ....................................................................................................................... 84 Figure 5.3. MALDI-TOF and SDS-PAGE gel of tOBP (A) and OBP::GQ20::SP-DS3 (B). Theoretical mass and the m/z values are indicated in (C). In (D) is shown the original gel from that was grouping the gels indicated in (A) and (B), being the truncated OBP the run of the line 2 and the OBP::GQ20::SP-DS3 the run of the line 4. ..................................................................................................................... 90 Figure 5.4. Circular dichroism (CD) spectra of wild-type OBP, truncated OBP (tOBP) and OBP::GQ20::SPDS3, at 25 and 37 °C. Final spectra were generated by the average of three scans for each sample. .................................................................................................................................................... 91 Figure 5.5. Structure of engineered OBP proteins analyzed by circular dichroism spectroscopy and molecular dynamics simulations. Secondary structure spectra determined by circular dichroism (CD) spectroscopy of tOBP (A) and OBP::GQ20::SP-DS3 (D); Top (B) and side (C) views of tOBP superimposed central structure; Top (E) and side (F) views of OBP::GQ20::SP-DS3 superimposed central structure. For all images, grey represents the structures at 25 °C, cyan and magenta represent the structures at 37 °C for tOBP and OBP::GQ20::SP-DS3, respectively. Percentage of secondary structures calculated from DSSP193 (Dictionary of Secondary Structure in Proteins) method implemented on GROMACS124, for the central structures (CS) (G). ...................................................................................................... 93 Figure 5.6. Backbone RMSD for wild-type OBP (A) along 60 ns MD simulation and for the engineered OBPs, along 20 ns of simulation time (B). Backbone atoms were fitted in all cases. ....................... 95 Figure 5.7. Binding curves of tOBP and OBP::GQ20::SP-DS3 obtained by measuring the fluorescence of 1 μM protein in 50 mM Tris–HCl, pH 7.5, at equilibrium with several concentrations of 1aminoanthracene (1-AMA). The dissociation constants were obtained at two temperatures, 25 and 37 °C, by mathematical fitting of data. Values are the mean ± SD of 3 independent experiments. ....... 96 Figure 5.8. Dissociation constants and binding pocket size for tOBP and OBP::GQ20::SP-DS3. (A) Dissociation constants and binding pocket size of tOBP and OBP::GQ20::SP-DS3 at 25 and 37 °C. [a]experimental data; [b]molecular simulation data. Schematic presentation of tOBP (B, C) and OBP::GQ20::SP-DS3 (D, E) binding pocket measured at 25 and 37 °C. Protein binding pocket size was
xviii calculated between the center of mass of opposite residues in the β-barrel: Tyr52 and Ile100 highlighted in blue sticks, considering the central structures obtained from the last 15 ns MD simulations. Values are the mean ± SD of 2 independent experiments. .......................................... 97 Figure 5.9. Fluorescence-binding assay of 1-AMA to tOBP and OBP::GQ20::SP-DS3. (A) Experimental layout for competitive binding evaluation; (B) visualization of 1-AMA in each compartment after variation of temperature; (C) amount of 1-AMA in each compartment after variation of temperature. Values are the mean ± SD of 2 independent experiments. .............................................................................. 99 Figure 5.10. Interaction bind mode of 1-AMA to OBPs estimated through molecular docking with AutoDock Vina. 1-AMA interaction with tOBP at 25 °C (A) and at 37 °C (B). Ligand bind mode of 1AMA to OBP::GQ20::SP-DS3 at 25 °C (C) and at 37 °C (D). 1-AMA ligand is presented in blue spheres and the amino acids involved in hydrogen bonds or van der Waals contacts, in CPK sticks representation. ........................................................................................................................... 100 Figure 5.11. Comparison between docked position and middle structure from MD simulations. The position of 1-AMA was estimated through molecular docking (1-AMA in cyan spheres) and through MD simulation (1-AMA in green spheres). A and B display the cavities estimated with PyMOL, in blue surface, for tOBP at 25 and 37 °C (A) and OBP::GQ20::SP-DS3, at 25 and 37 °C (B). In C is presented the most probable position for 1-AMA interaction with tOBP, at 25 and 37 °C, respectively. In D is shown the most probable bind mode of 1-AMA to OBP::GQ20::SP-DS3, at 25 and 37 °C, respectively. .................................................................................................................................................. 103 Figure 5.12. Binding energy along time obtained from g_mmpbsa tool. Running average binding curves in kJ/mol, of 1-AMA/tOBP complexes in grey and cyan, at 25 and 37 °C respectively and for 1AMA::OBP::GQ20/SP-DS3 complexes in black and mage\nta, at 25 and 37 °C respectively. ........ 104 Figure 6.1. Schematic presentation of β-citronellol release from functionalized fabrics. ................ 117 Figure 6.2. Evaluation of protein and fragrance loss during ethanol injection method for production of the CBM::GQ20::SP-DS3-liposome complex by SDS-PAGE electrophoresis (A): (1) CBM::GQ20::SP-DS3; (2) CBM::GQ20::SP-DS3-liposomes; (3) nonanchored protein (CBM::GQ20::SP-DS3); (Mw) 5 μL GRS Unstained Protein Marker (GRISP); (B) GC-MS spectrum of nonencapsulated β-citronellol when separated from liposomes encapsulating fragrance and anchoring protein though a membrane with 100 kDa cut-off. .......................................................................................................................... 118
xix Figure 6.3. Physicochemical characterization of nonfunctionalized liposomes (A, B) and functionalized liposomes encapsulating β-citronellol (C, D). Values represent the mean ± SD of 3 independent experiments................................................................................................................................ 119 Figure 6.4. Particles size (nm) and concentration (particles/mL) after 1 h incubation at 37 °C of nonfunctionalized and functionalized liposomes with an acidic sweat solution (pH 4.3 ± 0.2) (A); Graphical representation of concentration of particles versus size of particles in presence of buffer and acid sweat solution (B); for a better visualization of graphic for functionalized liposomes, different scales were used (C). ............................................................................................................................ 120 Figure 6.5. Amount of OBP::GQ20::CBM/β-citronellol and CBM::GQ20::SP-DS3-liposome/β-citronellol complex functionalized on cotton (%) determined by K/S evaluation. The values were obtained by subtracting the K/S value of buffer to the K/S value of each protein concentration....................... 124 Figure 6.6. SEM photographs of cotton fabrics functionalized with OBP::GQ20::CBM (A) and CBM::GQ20::SP-DS3-liposome (B) and control (C). In D is presented the quantification of the elements present in cotton functionalized with OBP::GQ20::CBM and CBM::GQ20::SP-DS3-liposome complex and control by energy-dispersive X-ray spectroscopy (EDS) analysis. The data are expressed in either weight or atomic concentration. SEM images magnification ranging from 3,900x to 4,300x; scale bar of 20 µm. *Phosphor content in EDS analysis is above 1000 ppm (statistically not reliable).221 ............... 125 Figure 6.7. Calibration curves of β-citronellol at 37 °C for 0.5 h (A) and 2 h (B) of SPME exposition time. .......................................................................................................................................... 126 Figure 6.8. β-citronellol chromatograms (RT = 16.4 min) of increasing concentrations of fragrance (A) and mass spectra of β-citronellol (B), observed by Headspace-SPME/GC-MS. ............................. 126 Figure 6.9. GC-MS chromatograms of cotton (A), nonfunctionalized liposomes (B) and acidic sweat solution (C). ................................................................................................................................ 127 Figure 6.10. β-citronellol release from cotton functionalized with OBP::GQ20::CBM/β-citronellol (green line) and with CBM::GQ20::SP-DS3-liposome/β-citronellol (grey line), after 1.5, 3, 8, 16 and 24 h of acidic sweat solution (pH 4.3 ± 0.2) exposure at 37 °C. The initial amount of β-citronellol added was 40 μM when using the OBP::GQ20::CBM, and 1000 μM when using the CBM::GQ20::SP-DS3liposome/β-citronellol. ................................................................................................................ 129
xx Figure 7.1. Applications of pig odorant-binding protein when conjugated with liposomes. (A) 1aminoanthracene transduction by OBP fused with cell-penetrating peptides (OBP::CPPs) and by (B) OBP fused with anchor peptide SP-DS3, with or without GQ20 spacer (OBP::SP-DS3 and OBP::GQ20::SPDS3). These approaches can be used for the capture of molecules, e.g. the capture of unpleasant or contaminant molecules. Figures based on/retrieved from Goncalves et al., 2018b; Gonçalves et al., 2018. ......................................................................................................................................... 132 Figure 7.2. Opposite binding behavior of truncated OBP (tOBP) and OBP::GQ20::SP-DS3. The engineered pig OBPs were separated in a beaker by a dialysis membrane with a cut-off permeable to 1-AMA (3.5 kDa). tOBP was placed inside the dialysis tube and the OBP::GQ20::SP-DS3 was placed outside. After equilibrating the 1-AMA concentration in both compartments, the proteins were added and the temperature was alternated between 25 and 37 °C. The binding in both compartments was measured for each temperature. The figure is based on Gonçalves et al., 2018a. ........................ 133 Figure 7.3. Fragrance release from OBP::GQ20::CBM functionalized on cotton, against perspiration. The release of fragrance was measured by GC-MS. Figure was retrieved from Goncalves et al., 2019. 135
xxi List of tables The tables were captioned considering that the first number refers to the chapter number. Table 2.1. Dissociation constants (Kd) of odorant molecules (ligands) for mammalian OBPs from different origin. The constant values are indicated for different conditions of binding experiment. The table was organized per alphabetic order of ligands for each mammalian OBP. .............................. 16 Table 2.2. Residues involved in the binding process of pig OBP and 1-aminoanthracene (1-AMA), undecanal (UND), benzophenone (BZP), benzyl benzoate (BZB), thymol and benzene ligands. The residues identification was performed through site-directed mutagenesis and molecular dynamics simulation. ................................................................................................................................... 28 Table 2.3. Residues involved in the binding process of bovine OBP and 2-amino-4-butyl-5propylselenazole, 5-propylselenazole, carvone, pyrazine, linalool, pyrazine2-isobutyl-3-methoxypyrazine (IBMP), dihydromyrcenol (DHM) and benzene. Residues identification was performed through site-directed mutagenesis and molecular dynamics simulation. ...................................................... 29 Table 2.4. Residues of human OBP (hOBPIIa) involved in the binding process of ligands like aldehydes or small carboxylic acids (undecanal (UND), N-phenyl-1-naphthylamine (1-NPN) and 11-(5- (dimethylaminonaphthalenyl-1-sulfonyl)-amino) undecanoic acid (DAUDA)). Residues of giant panda OBP (AimelOBP3) involved in the binding process of linear and long-chain aldehyde ligands. Residues identification was performed through site-directed mutagenesis and molecular dynamics simulation. .................................................................................................................................................... 30 Table 2.5. Residues involved in the binding process of buffalo OBP and 1-AMA, 1-octen-3-ol and oleic acid, p-cresol and undecanal (UND) ligands. The residues identification was performed through molecular dynamics simulation. .................................................................................................... 31 Table 3.1. Amino acid sequence, molecular weight and ionic charge of CPPs ................................ 52 Table 3.2. 1-AMA transduction efficiency driven by OBP::CPPs into liposomes and 1-AMA binding dissociation constants (Kd) of OBP::CPPs. The CPP effect was determined relatively to the OBP wt. Values are the mean ± SD of 2 independent experiments .............................................................. 56 Table 3.3. Physicochemical characterization of liposomes before and after 1-AMA transduction ..... 57 Table 3.4. 1-AMA distribution after peak deconvolution .................................................................. 59
xxii Table 4.1. Dissociations constant (KdF) for (R)-(-)-carvone 98% and (S)-(+)-carvone 96% fragrances determined by competitive binding 73 Table 5.1. Amino acid residues of tOBP (tOBP) and OBP::GQ20::SP-DS3 involved in the 1-AMA binding. The analysis was performed using the AutoDock Vina at 25 and 37 °C. In bold are described the new residues involved in the binding of 1-AMA to OBPs yet not identified in literature .......................... 102 Table 6.1. Association constants (Ka) of OBP::GQ20::CBM for 1-AMA (ligand model) and four fragrances (β-citronellol, coumarin, vanillin and eugenol) at 37 °C, at pH 7.5 for 1 h. ................................... 117 Table 6.2. CBM-based complexes characterizationa ..................................................................... 122
xxiii Abbreviations list 1-AMA: 1-aminoanthracene 1-NNN: N-phenylnaphthalen-1-amine 2-NNN: 2-naphthylamine 1-NPN: N-phenyl-1-naphthylamine or N-phenylnaphthalen-1-amine AC: adenylyl cyclase ASA: (±)-12-(9-anthroyloxy)stearic acid ATP: adenine triphosphate BSA: bovine serum albumin BZB: benzyl-benzoate BZP: benzophenone Ca2+: calcium ions cAMP: cyclic adenine monophosphate CBM: carbohydrate-binding module CD: circular dichroism spectroscopy CFP: cyan fluorescent protein Cl-: chloride ions CLSM: confocal laser scanning microscope CPP: cell-penetrating peptides CS: central structure DACA: dansyl-DL-α-aminocaprylic acid DAUCA: 11-((5-dimethylaminonaphthalenyl-1-sulfonyl)amino)undecanoic acid DHM: Dihydromyrcenol or 2,6-dimethyl-7-octen-2-ol DLS: Dynamic Light Scattering DOPE: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DSPE-PEG: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] DSSP: Dictionary of Secondary Structure in Proteins method EDS: Energy-dispersive X-ray spectroscopy
4 Chapter 2 Mammalian odorant-binding proteins
5 Published paper Filipa Goncalves, Artur Ribeiro, Carla Silva and Artur Cavaco-Paulo. Biotechnological applications of mammalian odorant-binding proteins. Critical Reviews in Biotechnology IBTY. DOI: 10.1080/07388551.2020.1853672 1. Introduction The olfactory system of mammals belongs to the chemical senses (smell, taste) playing a crucial role on the detection and presentation of different odorant molecules from the environment, translating them into varied perceptions and behaviors. This system includes odorant-binding proteins (OBPs), for the binding and presentation of odors, and olfactory receptors (ORs) located in olfactory sensory neurons, associated with the olfactory bulb, for the detection and discrimination of the odors.5-6 2. Mammalian olfactory transduction system The olfactory system of mammals has a vital importance in the identification of odorants; in prey/predators’ relations; for reproduction purposes and in the identification of toxic food.7-9 Olfactory perception is initiated when an odorant, existent in the air, is presented and interacts with the olfactory receptors (ORs) of the olfactory sensory neurons, through the nasal mucus of vertebrates.6, 10-11 This smelling process relays in a synergistic mechanism between the odorant, the odorant-binding proteins (OBPs) and the olfactory receptors (ORs).7, 11 The odorants are usually low molecular weight, hydrophobic and volatile molecules that cross the hydrophilic nasal mucus to reach the ORs in a process mediated by the OBPs.8, 11 The OBP/odorant complex is recognized by the OR, a protein belonging to the subfamily class A of G protein-coupled receptor (GPCR), existent in the olfactory sensory neurons.12 The trigger of OR lead to an intracellular signaling cascade resulting in the production of guanosine diphosphate (GDP) on the α subunit of the G-protein. The activated α subunit interacts with the adenylyl cyclase (AC) that converts adenine triphosphate (ATP) into cyclic adenine monophosphate (cAMP). This signaling cascade induces the inflow of Ca2+ and Na+ ions by activating the membrane channels. The consequent increase in the internal concentration of Ca2+ causes the opening of Ca2+-activated Clchannels that produces an efflux of Clfrom the neuronal cilia, contributing to the olfactory neuron membrane potential depolarization. The depolarization leads to action potentials
6 that are transmitted along the axon of the olfactory sensory neuron until reach the olfactory bulb followed by interpretation of olfactory signal in different areas of the brain (piriform cortex, olfactory tubercle, anterior olfactory nucleus, and specific parts of the amygdala and entorhinal cortex) (Figure 2.1).5-6, 13-15 Figure 2.1. Mechanism of mammalian olfactory system. In the presence of an odorant molecule (1), the odorant-binding proteins (OBPs) present in the nasal mucus carry it to the olfactory receptors (ORs) present in olfactory neuronal cell (2). Consequently, an intracellular signaling cascade is trigged via Gprotein (3), resulting in the production of cyclic nucleotide (cAMP) by the activation of adenylyl cyclase (AC) (4). cAMP induces the inflow of Ca2+ and Na+ ions which increase inside of neuronal cell causes an efflux of Cl- (5), contributing to the olfactory neuron depolarization. The depolarization of the olfactory neuron membrane leads to modification in action potential that is conducted along the axon until achieve the olfactory bulb and the olfactory signal is interpreted by the brain (6). Figure was created based on Sankaran et al., 201216 and Yoo et al., 201717. 3. The role of mammalian odorant-binding proteins The odorant-binding proteins (OBPs) are small (≈ 15-20 kDa), extracellular, water-soluble proteins and members of the lipocalins superfamily.18-21 OBPs are expressed in high concentration in the glands of the nasal mucosa and in the vomeronasal organ being released into the nasal mucus of vertebrates.3,
7 16, 20 Their expression occurs momentarily after birth, reaching their maximum levels within 2-3 days.22 During life-time, the OBPs are not produced at a constant rate, they are only expressed in certain periods and under certain physiological conditions.3 The first vertebrate OBP identified was isolated in 1985, from the nasal mucosa of bovines.23 In the last three decades, OBPs from other species, including pig24, rabbit25, elephant26, mouse27, rat28, insects29, frog30 and human31, have been also identified. Although the exact function of OBPs remains unclear, studies have been postulating that these proteins are involved in several processes related with the binding and presentation of odors. Once OBPs are secreted into the nasal mucous and present capacity to bind odors, it is believed that they are responsible to transport hydrophobic odorant molecules, in their calyx-shaped cavity, across the aqueous mucus barrier towards the olfactory receptors.5, 9, 21, 32-34 They also might be involved in the termination of the olfactory signal by removing odorants from the olfactory receptors after stimulation.9, 35-36 Furthermore and according to Ikematsu et al. (2005), OBPs can be also involved in general defense mechanisms in mammals, especially on the removal of harmful substances present in breathed air, thus maintaining the receptor binding sites in a state of readiness.37 Grolli et al. (2006) investigated the binding properties of OBPs to the 4-hydroxy-2-nonenal (HNE), a reactive lipid peroxidation end-product. With this study, they intended to establish a functional relation between the OBPs and the molecular mechanisms involved in the combat to free radical cellular damage. HNE is produced through the peroxidation of unsaturated fatty acids by cells as a consequence of the exposure to the oxygen present in the inhaled air. The binding data revealed a dissociation constants (Kd) of 4.9 and 9.0 μM for porcine and bovine OBP, respectively.38 This constant evaluates the strength of the interactions between the ligands and the protein. Low Kd values are observed by steeper slopes (fluorescence vs ligand concentration) and correspond to the greater binding affinity of the ligand to OBP; high Kd values mean weakly ligand affinity. These preliminary results suggest that OBP can be also associated with the reduction of HNE toxicity in the nasal mucosa.38 This discovery helps to understand the function of the OBP in the protection of the nasal mucosa, exposed to airflow and associated oxidative stress. Some authors referred that ORs have the ability to be stimulated by odors, even in the absence of OBPs.39-40 However, the results did not explain why the presence of OBP improved the affinity of OBP/odor complex by the receptor. Vidic et al. (2008) demonstrated that high concentration of helional odorant, an aldehyde used as a perfume in soap and laundry detergents, when in the absence of native rat
8 OBP-1 resulted in significantly diminution of the response of OR17-40 to helional.41 Furthermore, when the ligand concentration is high, OBPs are crucial to prevent the saturation of ORs binding sites.33, 41-43 Figure 2.2 resumes the main functions of mammalian OBPs and some of the organisms from where they were identified. Figure 2.2. Mammalian OBPs origin (left) and their functions in the olfactory system (right). Figure was created based on Gomez-Velasco et al., 202021; Grolli et al., 200638; Ikematsu et al., 200537; Muthukumar et al., 20189; Pelosi et al., 201433. Some OBP isoforms have been identified within the same species. These isoforms diverge in their amino acid sequences and in their specificity to different types of ligands.44 For example, there are several OBP isoforms in rat which differ in their binding preference.45 Rat OBP-1 preferentially binds to heterocyclic compounds, such as pyrazine derivatives, whereas OBP-2 presents more specificity for carboxylic acids and long-chain aliphatic aldehydes. On the other hand, rat OBP-3 appears to have a strong interaction with odorants composed by saturated or unsaturated ring structures.44-45 The OBPs from different species share low sequence similarity. For example, pig OBP-I (pOBP) and human OBP (hOBPIIa) share only 13.9% of sequence similarity, while pOBP and bovine OBP (bOBP) share 42.7%.32, 46 Intra OBP species analysis, e.g. testing two human OBPs sequences (hOBPIIa and hOBPIIb) displayed 95% of identity47, despite that hOBPIIa is expressed in the nasal mucus, saliva and, lachrymal glands; while hOBPIIb is expressed in the genital organs. A low number of 3D structures of
9 OBP proteins are available, as we confirmed in Protein Data Bank (PDB). Regarding the protein sequences predicted as OBPs, through blast analysis, we retrieved several sequences from PDB, UniProt (Universal Protein) and Ensembl genome browser.48 In order to better understand the similarities between OBPs from different species, a cladogram was created (Figure 2.3). Since this thesis is focused on mammalian OBPs and due to the high number of sequences observed (less sensitivity and low similarity), the sequences of birds, reptiles and bony fishes were rejected. All different sequences, even from the same species, were considered to construct the cladogram. The results show that all mammalian species are well distributed in different orders. Figure 2.3. Cladogram of mammalian OBPs. The protein sequences available for mammalian OBPs were acquired from PDB, Uniprot and Ensembl databases. Using the MUSCLE software 95 sequences were aligned, and the spurious sequences or poorly aligned regions from a multiple sequence alignment were removed through the trimAl tool. The sequences were aligned, and phylogenetic tree
10 accessed using the IQ-TREE Web Server selecting the use of 1000 number of bootstrap alignments, and the management of the tree was obtained through iTOL online tool. Different orders are represented at different colors: rodentia (light grey), artiodactyla (ligth green), carnivora (dark blue), chiroptera (dark grey), cingulata (ligth blue), dasyuromorphia (lilac), didelphimorphia (purple), diprotodontia (orange), erinaceomorpha (yellow), lagomorpha (dark pink), perissodactyla (dark green), primates (red), proboscidea (ligth pink) and rodentia (ligth grey). 4. Physicochemical and structural properties of OBPs In the last two decades several structures of OBPs have been solved by X-ray diffraction and deposited in Protein Data Bank (PDB). However, a low number of 3D structures are available. In 1996, a OBP structure isolated from bovine ( Bos taurus ) nasal mucosa at 2.0 Å of resolution (PDB ID: 1OBP) was reported.49 Spinelli et al. (1998) obtained the crystallographic structure of pig ( Sus scrofa ) OBP with 2.25 Å of resolution (PDB ID: 1A3Y).50 Actually, there is a more accurate structure of pOBP with 1.48 Å of resolution (PDB ID: 1DZK).51 Also, White et al. (2009) reported the crystal structure of rat OBP-1 at 1.6 Å resolution (PDB ID: 3FIQ) and the monomeric state of this protein, either in the crystalline form or in solution, when under native conditions.43 Crystal structure of the human OBP, designated as OBPIIa, was also obtained (PDB ID: 4RUN).52 The protein sequences of these OBPs are very different, as we can observe in the alignment of the sequences at figure 2.4A. The structures of the OBPs available in PDB are presented in figure 2.4B. Despite many other OBP sequences are known (total or partially) their structure is not yet resolved.
11 Figure 2.4. Mammalian OBP protein sequences (A) and structures (B). The multiple alignment of the OBP sequences, with structure resolved in Protein Data Bank (PDB), was performed using the MUSCLE software (A); an ‘*’ (asterisk) indicates the positions which have a single, fully conserved residue, ‘:’ (colon) indicates conservation between residues belong to groups of strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix), a ‘.’ (period) indicates conservation between groups of weakly similar properties (scoring = < 0.5 in the Gonnet PAM 250 matrix). The OBP structures observed in (B) were retrieved from PDB. In the figure are presented the OBPs from: human Homo sapiens OBPIIa (PDB ID: 4RUN), boar Sus scrofa (1GM6); giant panda Ailuropoda melanoleuca (5NGH); rat Rattus norvegicus OBP (3ZQ3); pig OBP isolated from Sus scrofa (1DZK); OBP from bovine Bos taurus (1OBP); and an isoform of OBP-1 from rat Rattus norvegicus (3FIQ). The primate order is represented by red box, the artiodactyla order is represented at light green box, the carnivora order is represented by dark blue box and the rodentia order is represented by light grey box. Despite the genetic variability between OBPs from different mammalian species, lipocalin members present some characteristics that allow their identification. For example, their tertiary structure is well conserved, with the β-barrel structure composed by eight β-strands (designed by A to H) linked by seven loops (L1 to L7) and connected to a short α-helix close to the C-terminus and a ninth β-strand followed by the disordered C-terminal tail.50, 53-54 Fourier-transform infrared spectroscopy (FT-IR)
12 spectrum of pig OBP (pOBP) suggested the existence of 51% β-sheets and 8% α-helix corroborating the values observed by X-ray diffraction (43% and 10%, respectively).55 Vertebrate OBPs have other conserved characteristics that allow their identification: a GxW motif (G: glycine; x can be any residue; W: tryptophan) on the N-terminus (residues 14-16, in pOBP); a glycine residue at the C-terminus (Gly119, in pOBP); and two cysteines in the middle and at the C-terminal end of the sequence.46 In porcine, the conserved Cys63, and Cys155, form a disulfide bridge tightening the flexible C-terminal α-helix domain and the β-barrel (Figure 2.5). In addition, an YxxxYxG motif can also be found (residues 78-84, in pOBP). Negatively charged residues (Glu and Asp) are also systematically present at positions 46, 130, 143 and 153 in pOBP. In OBPs, except for Asn145, there are some aromatic and aliphatic residues or glycine completely conserved (Phe55, Phe88, Phe132, Ile104, Ile141, Ile146, Gly109, Gly119, Gly140 in pOBP).32 Figure 2.5. Cartoon diagram of porcine OBP protein (pOBP). The figure indicates the localization of Trp16 residue (red, W16), five Tyr residues (blue, Y20, Y52, Y78, Y82 and Y92), and the disulfide bridge between Cys63 and Cys155 (yellow). The figure was retrieved from Staiano et al., 200756, constructed on the basis of pOBP structure given in file 1A3Y.pdb. Bovine OBP (bOBP) differs from the other lipocalins by lacking the conserved disulfide bond and by their ability to form domain-swapped dimers.57 bOBP was the first mammalian OBP with crystal structure solved and the analysis of this structure revealed a domain-swapped dimer, in which the helix
13 near the C-terminal region of each monomer is packed against the β-barrel of the other.49, 58 The domain swapping mechanism of this OBP was explained by the absence of a glycine residue at the hinge region linking the β-barrel to the α-helix and also by the lack of the disulfide bridge.59 A bovine mutant OBP obtained by the insertion of a glycine residue in the position 121 of bOBP, and by the replacement of W64C and H155C (GCC-bOBP), resulted in a monomeric protein without any structure perturbation revealing the importance of the absence of the Gly121 and cysteine residues during the formation of the dimeric structure.57 Experimental and computational data also revealed that bOBP is a dimer at neutral pH, contradicting the monomeric structure at pH 2.5. Acid pH results in the loss of the swapped dimeric conformation, without alteration of the tertiary and secondary structures.60 The structure of mammalian OBPs proved to be highly stable and resistant to degradation by temperature, organic solvents, pH variation, or proteolytic digestion.33-34 Vertebrate OBPs can resist at high temperatures before undergoing denaturation, and, if unfolding occurs, this phenomenon can be reversed after restoring the initial conditions.55 The FT-IR spectra for pOBP revealed a structure exceptionally stable to thermal denaturation (up to 80 °C), particularly in the presence of the 2isobuthyl-3-methoxypyrazine and 3,7-dimethyl-1-octanol ligands.55 The FT-IR data also showed two transition phases occurring at 65–70 °C and 80–85 °C, related to molten globule states of the βbarrel, maintaining however the structural integrity at such temperatures.55 Besides, circular dichroism spectroscopy measurements proved that the pOBP preserves a structural stability up to 65 °C.61 Exceptionally, bOBP maintains the dimer form even in the presence of 1.5 M of guanidinium chloride (GdnHCl).62 Also pOBP is only completely unfolded when dissolved in solutions containing until 3.5 M of (GdnHCl)56 or when suspended in 8 M of urea, as verified by circular dichroism spectroscopy.63 5. Binding affinity and selectivity of mammalian OBPs The first mammalian OBP was discovered in the nasal mucosa of bovines, by ligand-binding experiments using a radioactive pyrazine ligand ([3H]-2-isobutyl-3-methoxypyrazine).23, 64 The first OBPs binding assays were based on methods involving radioactive-labelled ligands and including the separation of bound and free ligand by electrophoresis, gel filtrations or others techniques.33, 64-65 These ligand-binding experiments, used to measure the affinity of odorants towards OBPs, were improved over time, replacing the radioactive probes by fluorescent ligands.65 Three approaches can be used to measure the binding of a ligand to OBP. The first is based on centrifugation steps where the free ligand
20 Dissociation constant (Kd) μM OBP origin Ligand Binding conditions References 0.38 Mutant pOBP-F88W Citralva 2 μM OBP pH 7.4 + 2 μM 1-NNN + 0.1-2 μM competitor, 25 °C, Ex. 345 nm, Em. 412 nm 4 0.12 Decanal 4 0.14 Diphenyl 1 μM protein pH 7.4 + 1 μM 1-AMA + 1-5 μM competitor, 25 °C, Ex. 295 nm 71 0.50 Fluoranthene 71 0.65 Fluorene 71 0.10 Geosmin 1 μM protein pH 7.4 + 1 μM 1-AMA, 2-20 μM competitor, Em. 480 nm 75 0.14 2-isobutyl-3-metoxypyrazine (IBMP) 75 1.00 2-naphthylamine (2-NNN) 2 μM OBP pH 7.4 + 0-2 μM synthetic ligand, 25 °C, Ex. 345 nm, Em. 412 nm 4 0.26 N-phenylnaphthalen-1-amine (1-NNN) 4 0.27 Phenanthrene 1 μM protein pH 7.4 + 1 μM 1-AMA + 1-5 μM competitor, 25 °C, Ex. 295 nm 71 0.48 9-phenylanthracene 0.93 Phthalazine 0.18 Pyrene
21 Dissociation constant (Kd) μM OBP origin Ligand Binding conditions References 0.50 Mutant pOBP-F88W Synthetic phenyl amine (PAA) 2 μM OBP pH 7.4 + 0-2 μM synthetic ligand, 25 °C, Ex. 410 nm, Em. 510 nm 4 0.17 Synthetic phenyl amine (PAF) 2 μM OBP pH 7.4 + 0-2 μM synthetic ligand, 25 °C, Ex. 315 nm, Em. 515 nm 4 0.23 Bovine wild-type OBP (bOBPwt) 1-aminoanthracene (1-AMA) 2 μM OBP pH 7.8 + 0.1-5 μM 1-AMA, 24 h, Ex. 360 nm, Em. 484 nm 60 1.52 2 μM OBP pH 2.5 + 0.1-5 μM 1-AMA, 24 h, Ex. 360 nm, Em. 484 nm 60 1.00 1 μM OBP + 0.076-5 µM 1-AMA, Ex. 380 nm, Em. 480 nm 70 0.80 Benzophenone (BZP) 0.5 μM OBP + 3 μM 1-AMA + 0.39-50 μM competitor, Ex. 380 nm, Em. 480 nm 70 0.35 Dihydromyrcenol (DHM) 0.5 μM OBP + 3 μM 1-AMA + 0.39-50 μM competitor, Ex. 380 nm, Em. 480 nm 70 9.00 4-hydroxy-2-nonenal (HNE) 1 μM OBP pH 7.8 + 1.5 μM 1-AMA, RT 20 min + 1-70 µM competitor, RT, 30 min, Ex. 380 nm, Em. 480 nm 38
22 Dissociation constant (Kd) μM OBP origin Ligand Binding conditions References 3.30 Bovine wild-type OBP (bOBPwt) 2-isobutyl-3-metoxypyrazine (IBMP) 0.5 μM OBP + 3 μM 1-AMA + 0.39-50 μM competitor, Ex. 380 nm, Em. 480 nm 70 1.20 1-octen-3-ol (OCT) 70 3.30 1-octen-3-ol (OCT) 0.76 μM OBP pH 8.5 + 2 μM 1-AMA + 0.39-50 μM competitor, 24h, 4 °C, Ex. 380 nm, Em. 480 nm 77 0.30 Undecanal (UND) 0.5 μM OBP + 3 μM 1-AMA + 0.39-50 μM competitor, Ex. 380 nm, Em. 480 nm 70 5.00 Mutant GCC-bOBP 1-aminoanthracene (1-AMA) 5 μM protein pH 7.0 + 0-50 μM 1-AMA, Ex. 295 nm, Em. 487 nm 78 0.66 Mutant bovine OBP 1-aminoanthracene (1-AMA) 0.75 μM protein pH 7.8 + 0.1-8 μM 1-AMA, 48 h, 4 °C, Ex. 380 nm, Em. 480 nm 59 3.80 Rabbit wild-type OBP (rabOBPwt) N-phenylnaphthalen-1-amine (1-NPN) 2 μM protein + 2-16 μM 1-NPN Ex. 337 nm, Em. 415 nm 79 7.80 2-nonenal 4 μM protein + 4 μM 1-NPN + 0-16 μM competitor, Ex. 337 nm, Em. 415 nm 79 11.20 Geraniol 79 2.20 Quercetin 79
23 Dissociation constant (Kd) OBP origin Ligand Binding conditions References 0.97 Human wild-tpye OBP (hOBPIIa) (±)-12-(9-anthroyloxy)stearic acid (ASA) 2 μM protein pH 7.5 + 5 μM 1-NPN + 0-30 μM ASA, 1 min, Ex. 360 nm, Em. 425 nm 31 8.10 dansyl-DL-α-aminocaprylic acid (DACA) 2 μM protein pH 7.5 + 5 μM 1-NPN + 0-30 μM DACA, 25 °C, 1 min, Ex. 345 nm, Em. 475 nm 31 1.50 11-((5-dimethylaminonaphthalenyl1-sulfonyl)amino)undecanoic acid (DAUCA) 2 μM protein pH 7.5 + 4 μM DAUCA, 25 °C, 1 min, Ex. 345 nm, Em. 490 nm 31 > 10.00 Eugenol 2 μM protein pH 7.5 + 5 μM 1-NPN + 0-30 μM competitor, 25 °C, 1 min, Ex. 337 nm, Em. 400 nm 31 0.50 Human wild-tpye OBP (hOBPIIa) Lilial 2 μM protein pH 7.5 + 5 μM 1-NPN + 0-30 μM competitor, 25 °C, 1 min, Ex. 337 nm, Em. 400 nm 31 2.00 Octanoic acid 31 0.30 Palmitic acid 31 0.30 Undecanal 31 1.00 Vanillin 2 μM protein pH 7.5 + 5 μM 1-NPN + 0-30 μM competitor, 25 °C, 1 min, Ex. 337 nm, Em. 400 nm 31
24 Dissociation constant (Kd) μM OBP origin Ligand Binding conditions References 3.30 Human wild-tpye OBP (hOBPIIa) N-phenylnaphthalen-1-amine (1-NPN) 2 μM protein pH 7.5 + 4 μM 1-NPN, 25 °C, 1 min, Ex. 337 nm, Em. 400 nm 31 2.24 2 μM protein pH 7.5 + 0-30 μM 1-NPN, pH 7.5, 25 °C, 3 min, Ex. 337, Em. 400 nm 80 3.27 Mutant hOBPIIa-K62A 2 μM protein pH 7.5 + 0-30 μM 1-NPN, pH 7.5, 25 °C, 3 min, Ex. 337, Em. 400 nm 80 3.01 Mutant hOBPIIa-K82A 80 6.78 Mutant hOBPIIa-K112A 80 0.21 Human wild-type OBP (hOBPIIa) Undecanal (UND) hOBP-NPN complex + undecanal, pH 7.5, 25 °C, 3 min, Ex. 337, Em. 400 nm 80 0.27 Mutant hOBPIIaK62A hOBP-NPN complex + undecanal, pH 7.5, 25 °C, 3 min, Ex. 337, Em. 400 nm 80 0.28 Mutant hOBPIIaK82A hOBP-NPN complex + undecanal, pH 7.5, 25 °C, 3 min, Ex. 337, Em. 400 nm 80 2.52 Mutant hOBPIIaK112A 80 2.91 Human wild-type OBP (hOBPIIa) Undecanoic acid (DAUCA) 2 μM protein pH 7.5 + 0-30 μM DAUCA, 25 °C, 3 min, Ex. 345, Em. 490 nm 80
25 Dissociation constant (Kd) μM OBP origin Ligand Binding conditions References 3.02 Mutant hOBPIIaK62A Undecanoic acid (DAUCA) 2 μM protein pH 7.5 + 0-30 μM DAUCA, 25 °C, 3 min, Ex. 345, Em. 490 nm 80 3.29 Mutant hOBPIIaK82A 80 3.42 Mutant hOBPIIaK112A 80
26 6. Mechanistic insights about OBP mode of action The formation of the OBP/ligand complex is dependent on the type of ligand, on the ligand proximity to the OBP protein, on the OBP structure, and the size of OBP cavity.4, 71 During complex formation, different amino acidic residues of OBP are involved in the binding process. Site-directed mutagenesis, recombinant technologies, advances in protein sequencing and the use of different bioinformatic tools, allowed the identification of the amino acids and the interactions involved in the binding process. Molecular dynamics (MD) simulations and docking analysis allow to calculate the coordinates of Cα atoms of protein variations; the thermodynamics and energies involved in binding; and to predict the structure of OBP with and without ligand. Furthermore, using MD simulations it is possible to infer and to quantify the percentages of secondary structure elements of OBPs; evaluate the solvent accessibility of amino acid residues; and identify the physicochemical interactions involved in the binding process. Together with site-directed mutagenesis, this tool helps to identify the residues involved in the OBP/ligand interaction.81-82 6.1. The nature of the ligand influences the binding site at OBP surface OBPs are known by their capacity to bind ligands with different sizes and functional groups. Several studies have been done to understand the binding process and to identify which residues on OBP’s binding site interact with the ligands during the formation of the OBP/ligand complex. While some ligands seem to interact with ‘universal’ residues of the OBP binding site, others bind in a more specific manner. The nature of the ligand seems to influence the binding process and to determine the amino acids of OBP involved in the binding. In pig OBP, the highly conserved Tyr82 residue has been identified as being involved in the binding process of several ligands. The replacement of this amino acid present in the protein binding pocket, by another residue, with the same physicochemical properties, like phenylalanine, resulted in any alteration on the cavity structure, which is consistent with the assigned function.83 Notwithstanding, the substitution of the same residue by an alanine (small hydrophobic amino acid), resulted in a structural alteration of the binding pocket, supporting the involvement of Tyr82 in the binding process.83 Meillour et al. (2009) demonstrated experimentally that Phe35 and Tyr82, both located in the pOBP pocket, are involved in the binding and release of 1-AMA (aromatic) and of undecanal (UND, aldehyde) ligands.84 The authors observed that the fluorescence spectra for the pOBP-Phe35Ala and pOBP-Tyr82Ala mutants, with increased concentrations of 1-AMA, presented
27 very similar fluorescent value, and different of wild-type pOBP. This result indicates that both aromatic residues, Phe35 and Tyr82, are necessary to retain the ligand in the binding pocket of pOBP. In the double mutant pOBP-Phe35Ala/Tyr82Ala, the maximum emission fluorescence did not shift upon 1AMA addition, indicating the absence of 1-AMA binding towards the OBP mutant.84 Molecular dynamic (MD) simulations supported the reported experimental data. The binding process is initiated by the opening of the OBP pocket site through the shift of tyrosine and phenylalanine residues.46 MD simulations report that the interaction between pig OBP and the ligand occurs through the shift of the residues mainly located at the junction between the β-strands D and E and the L1 and L5 loop (Figure 2.6).85 Figure 2.6. Cartoon illustration of pig OBP (PDB ID: 1DZK). The elements of the secondary structure are indicated (β-stands A-H, α-helix and Loops L1-L7). The figure was created using PyMOL. The number of interactions and the residues involved between OBP and the different odorants have been found to vary between two and ten residues. For example, in pOBP, the Ile21, Met39, Val80, Tyr82, Phe88, Ile100, Asn102, Met114, Gly116 and Leu118 residues are involved in the interaction with benzophenone (BZP, aromatic ketone).51 For benzyl benzoate (BZB) the residues involved in the binding are described to be Asn86, Phe88, Asn102, Met114 and Thr115.51, 86 The 2,6-dimethyl-7-octen2-ol (DHM), the undecanal (UND, aldehyde) and the 2-isobutyl-3-metoxypyrazine (IBMP) are oriented differently in the cavity of the protein and interact with different amino acid residues (Asn86, Asn102,
28 Try82, Phe35, Phe55, Lys28 and Asp110).51, 84-85 While for some ligands, several amino acid residues are involved in the ligand interaction, for others, few residues participate on the process. For example, the binding of thymol to pOBP seems to be mediated by only two residues, the non-polar Ile21 and Phe88.51 Table 2.2 presents the residues identified as being involved in the binding of wild-type pig OBP to the specific ligand. Table 2.2. Residues involved in the binding process of pig OBP and 1-aminoanthracene (1-AMA), undecanal (UND), benzophenone (BZP), benzyl benzoate (BZB), thymol and benzene ligands. The residues identification was performed through site-directed mutagenesis and molecular dynamics simulation. Pig OBP 1-AMA UND BZP BZB Thymol Benzene Ile21 ● ● ● Phe35 ● Met39 ● Phe35 ● ● Val80 ● Tyr82 ● ● ● Asn86 ● Phe88 ● ● ● Ile100 ● Asn102 ● ● ● Met114 ● ● ● Thr115 ● ● Gly116 ● ● Leu118 ● References 83 84 83 84 46 51 51, 86 51 61 Bianchet et al. showed that in bovine OBP (bOBP) the access of a ligand to the binding cavity of the protein is controlled by the hydrophobic aromatic Phe89, through rotation around a carbon–carbon single bond.58 Phe89 but also Phe54 seem to control the access to the bOBP pocket.32 Hajjar et al. (2006) showed, using MD simulation, that the hydrogen bonding between the aromatic polar Tyr21 and Tyr79 residues increase in the presence of the thymol ligand. Tyr83 (conserved Tyr82 in pOBP)
29 was also referred as being situated near the entrance of the binding cavity as demonstrated by MD simulations. This amino acid constitutes and regulates the entrance to the bOBP cavity.85, 87 Moreover, Tyr83 appears to be especially involved in the unbinding process and can be considered as the gate of OBPs’ binding pocket. This amino acid is highly conserved among the members of the lipocalin superfamily, indicating its high relevance on the binding process.85 Other interaction studies, revealed that Phe36 and Tyr83 residues are responsible to regulate the access to the binding site in bOBP, by the rotation of residues side chains, opening the barrel entrance and increasing the binding cavity volume.60 Table 2.3 presents the residues identified as being involved in the binding of some ligands to the bovine OBP. Table 2.3. Residues involved in the binding process of bovine OBP and 2-amino-4-butyl-5propylselenazole, 5-propylselenazole, carvone, pyrazine, linalool, pyrazine2-isobutyl-3-methoxypyrazine (IBMP), dihydromyrcenol (DHM) and benzene. Residues identification was performed through site-directed mutagenesis and molecular dynamics simulation. Bovine OBP 2-amino-4-butyl5-propylselenazole, carvone or pyrazine Linalool IBMP DHM Benzene Tyr21 ● Pro35 ● Phe36 ● Phe54 ● ● Tyr79 ● Tyr83 ● ● Phe89 ● ● ● Ala101 ● Asn103 ● Leu115 ● Thr116 ● Phe119 ● References 32 87 85 60 61 Studies with OBPs from other mammals have also been performed to understand which amino acids are involved in the binding process with different ligands. Human variant hOBPIIa presents affinity to
36 7.1.4. Biosensors for medical diagnosis Differentiated biological ligands can be applied as potential biomarkers for the detection of cancer and other diseases. The immobilization of a human OBP on a nanopore array and its response to docosahexaenoic acid, lauric acid and benzaldehyde, has been reported as a new biosensor with potential application in the field of medical diagnosis. In this study, the authors measured changes on electric impedance to detect these compounds.100 More recently, it was described the potential of OBPbased sensors to screen volatile organic chemicals (VOCs) emitted by organisms, as components of non-invasive medical procedures, to monitor a patient's metabolic state or diagnose pathological conditions.34 For example, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor was described to monitor and to quantify ethanol and other alcohols in living cells.101 The nanosensor was designed using a human OBP (hOBPIIa) flanked by two fluorescent proteins, enhanced cyan fluorescent protein (ECFP) and Venus at Cand N-terminus of OBP, respectively. The sensor revealed a dissociation constant (Kd) of 4.16 μM for ethanol and due to its characteristics is a noninvasive sensing device with potential to be used in bacteria, yeast and mammalian cells.101 7.2. OBPs as capture and release devices of odorant molecules OBPs showed to be ideal for the development of versatile sensing elements. Yet their applications, can be further extended to other areas, like the capture of unpleasant odors and the programmed release of fragrance molecules. In a study of Silva et al. (2014), the authors explored the use of pig OBP (pOBP) functionalized onto cotton fabrics for the release of fragrances, and the reduction of unpleasant odors, such as cigarette smoke.67 The authors evaluated the affinity of four fragrances (βcitronellol, citronellyl valerate, ethyl valerate and benzyl benzoate) to pOBP by competitive assay, with the β-citronellol presenting the highest affinity.67 After functionalization of cationized cotton fabrics (chemical reaction of cationic reactive agents with cellulose to impart positive charges to cotton surface) with the pOBP/citronellol complex (1:2 molar proportion), the release of β-citronellol was evaluated at 37 °C by headspace gas chromatography–mass spectrometry (HS-GC-MS). After 5 min at 37 °C, a release around 42% of the initial concentration of fragrance was detected.67 A human panel evaluated the cigarette smell in samples of cotton functionalized with pOBP and verified a higher reduction of the smoke smell when compared with non-functionalized cotton samples.67 This research opened the potential of OBPs as devices for the capture of odors and for the release of fragrances and
37 other molecules (antimicrobial agents and insect repellents), with high interest for the textile and cosmetic industries, as explored in this thesis. 8. Future Prospects Odor perception is a vital process for the animals in general. It helps the animals in the identification of members from the same or from different species, the identification of food and of potential poisons.79 The olfactory event involves the interactions of the odorants with the olfactory receptors assisted by the OBPs. Although the mechanisms of interaction between the OBP and ligands, and of the OBP/ligand complex with the ORs are still not well understood, it has been demonstrated that OBPs contribute to olfactory perception at several levels: transport, prevention of oxidative stress and prevention of ORs saturation. Mammalian OBPs act as passive carriers of hydrophobic odorant molecules from environment to the olfactory receptors.5, 13, 16, 87 The β-barrel structure of OBPs and their ability to bind different types of molecules, suggests their potential role in vertebrates olfactory system. The total knowledge of the olfactory system and the mechanisms involved in smell perception are still scarce, mostly due to the complexity of the mechanisms involved, from the molecular aspects of the odorant bindings to the final signal transduction to the limbic system. It is crucial to understand the molecular mechanisms involved in the association and dissociation between the ligand and the OBP; and between the OBP/ligand complex with the OR. Ligand-binding experiments allow characterizing OBPs, their role in chemical communication, and their capacity to associate/dissociate to different molecules. With the advances in the informatics tools, molecular dynamic simulations and molecular docking experiments have provided detailed information about the nature of OBP/ligand interactions and the position of ligands in the proteins. Genomics and proteomics have allowed to obtain more information about the identification and function of OBPs. These new tools can also help the identification of new OBPs and their function. For example, some binding proteins were identified in databases as OBPs but the main ligands are pheromones; thus, this proteins’ classification would be renamed as pheromone-binding protein. The outstanding stability of OBPs to thermal denaturation, pH and proteolytic degradation make these proteins excellent candidates for the development of sensing devices for pollution control, agriculture, healthcare, security, cosmetic and food industry applications.33-34, 93 Furthermore, OBP is involved in the smell perception and some studies suggest that it could be used for odor control and for the controlled
38 release of fragrances from a textile.67, 102 The utilization of OBPs in several reported fields are dependent on the social and economic impact, and on the advantages (solubility, stability, detection of different molecules) and disadvantages (low selectivity and sensitivity) associated.
39 Chapter 3 OBP fused with Cell-penetrating Peptides promotes Liposomal Transduction
40 OBP fused with Cell-penetrating Peptides promotes Liposomal Transduction Abstract Cell-penetrating peptides (CPPs) have been applied as novel transport systems with the ability to facilitate the delivery of peptides, proteins, and oligonucleotides into cells. Herein, we designed different fusion proteins composed by pig odorant binding protein (OBP-I) and three CPPs, namely Tat, pVEC and Pep-1. A new methodology using liposomes as reservoirs and OBP::CPPs as carriers was developed as an advanced system to capture odorant molecules. 1-aminoanthracene (1-AMA) was used as a model molecule to evaluate the transduction ability of OBP::CPPs into the reservoirs. The transduction efficiency was dependent on the initial capacity of OBP::CPPs to bind 1-AMA and on the penetration of liposomes promoted by the CPPs. An encapsulation efficiency of 42% was obtained with OBP::Tat fusion protein. The presence of Tat peptide increased the 1-AMA transduction of 1.3 and 2.5 fold compared with Pep-1 and pVEC, respectively. This work expands the application of OBPs and CPPs on the design of promising capture and delivery systems for textile and cosmetic applications. Published paper Filipa Gonçalves, Tarsila G. Castro, Eugénia Nogueira, Ricardo Pires, Carla Silva, Artur Ribeiro, Artur Cavaco-Paulo. Colloids and Surfaces B: Biointerfaces 161 (2018) 645–653. DOI: 10.1016/j.colsurfb.2017.11.026
41 1. Introduction CPPs are cationic peptides, normally up to 30 residues, which can be amphipathic or hydrophobic, possessing low cellular toxicity.103 These peptides are widely studied to deliver biologically active molecules into cells, such as peptides, proteins, RNA, DNA, oligonucleotides and liposomes without the need of specific membrane receptors.104-105 Several works report the use of CPPs in biomedical applications. Tat-conjugated quantum dots administrated intra-arterially at a proximal cervical carotid artery in rats were able to cross the highly impermeable blood-brain barrier.103 Elmquist et al. (2001) demonstrated the internalization of pVEC labeled with fluorescein isothiocyanate (FITC) into three different endothelial cell lines after treatment with this peptide.106 Jing et al. (2016) demonstrated that the combined use of CPPs-loaded nanobubbles with ultrasound-targeted microbubble destruction (UTMD) technology could efficiently improve gene transfection in cultured breast cancer TNBC cells.107 The application of CPPs in other areas rather than therapeutics, such as textile functionalization, was exploited herein for the first time. CPPs were conjugated with an odorant-binding protein (OBP) for the capture and transduction of odorant molecules. Liposomes were used as final reservoirs and 1-AMA as the model molecule. Odorant-binding protein I (PDB ID: 1DZK) is a transport protein present in the nasal mucosa of pig constituted by 157 amino acids. This protein was selected for this study based on the information available about the three-dimensional structure and the binding specificity for a large number of natural and synthetic molecules.4, 76 OBP-I is composed by eight antiparallel β-sheets108, forming an internal cavity to bind different ligands, like terpenoids, aromatic compounds, aliphatic molecules and aldehydes.4 OBPs have been studied in several applications. Wei et al. (2008) designed different mutants of pig OBP to bind several aromatic polycyclic hydrocarbons. This study opens the view for the use of OBPs as biosensors for the monitoring of aromatic pollutants.71 Di Pietrantonio et al. (2013) used surface acoustic wave (SAW) biosensor systems with three different OBPs as probes for the detection of low concentration of octanol (13 ppm) and carvone (9 ppm).92 More recently, Silva et al. (2014) used pig OBP for the reduction of unpleasant odors and controlled release of fragrances when immobilized in fabric supports.67 We designed for the first time three OBP::CPPs to be used as carriers and transducers of a model molecule, 1-aminoanthracene (1-AMA), as represented in Figure 3.1. The CPPs used in this work were Tat, pVEC and Pep-1. Tat corresponds to the basic domain of HIV-1 Tat protein rich in arginine
42 residues.109 pVEC is derived from the murine sequence of the cell adhesion molecule vascular endothelial cadherin with an amphipathic character106 and Pep-1 is a synthetic peptide that belongs to the group of amphipathic peptides. It contains a hydrophobic tryptophan-rich domain and a hydrophilic lysine-rich domain.110 1-AMA is the most used ligand to characterize the ligand binding properties of OBP family members.86, 111 When 1-AMA binds to OBP-I, the maximum wavelength of AMA/OBP complex is shifted from 537 to 481 nm, with an increase of fluorescence intensity compared with the ligand alone.67, 108 A new methodology was developed to evaluate the transport and transduction of 1-AMA into reservoirs promoted by OBP::CPPs. Figure 3.1. Cartoon representation of OBP wt and OBP fused with CPPs, complexed with 1-AMA. The OBP is shown in grey scale, 1-AMA in magenta and the CPPs in rainbow colors; the position of the ligand on each OBP system was settled through a standard molecular docking procedure with AutoDock4 and represents the best binding energy.
43 2. Materials and Methods 2.1. Reagents Tris-base, imidazole, sodium phosphate, sodium chloride, cholesterol and fluorescein isothiocyanate (FITC) were obtained from MerckSigma, Spain. 1-AMA was purchased from TCI chemicals, Belgium. Nickel Magnetic Beads for His Tag Protein Purification was available from Biotool, Bimake, Spain. Molecular weight Precision Plus ProteinTM standards were purchased from BioRad, Portugal. Culture medium was purchased from GRISP, Portugal. 1,2-Dioleoyl-sn-glycero-3phosphoethanolamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG) were purchased from Lipoid, Canada. Alexa Fluor 467 was purchased from ThermoFisher Scientific, France. All other reagents were acquired from MerckSigma and used as received. 2.2. OBP fusion constructs Three CPPs: Tat (GRKKRRQRRRPPQ), Pep-1 (KETWWETWWTEWSQPKKKRKV) and pVEC (LLIILRRRIRKQAHAHSK) were fused in the C-terminus of OBP-I. Gene sequences (OBP::Tat, OBP::Pep1 and OBP::pVEC) were synthetized by GenScript and cloned in pET-28a plasmid. 2.3. CPPs synthesis CPPs were synthetized by JPT peptide technologies GmbH with 94.7%, 98.0% and 98.2% of purity for Tat, Pep-1 and pVEC, respectively. These peptides were used as experiment controls. 2.4. Expression and purification of fusion OBPs Escherichia coli BL21(DE3) harboring the pET-28a:OBP::CPPs was used for protein expression in Terrific Broth (TB) medium supplemented with lactose. Cells were harvested by centrifugation at 7,000 g, for 5 min at 4 °C and resuspended in phosphate buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4) supplemented with 10 mM of imidazole and lysed by sonication (40%, 3.0 sec ON, 9.0 sec OFF for 10 min) in sonicator vibracellTM SONICS. Soluble and insoluble fractions were separated by centrifugation at 12,000 g, for 30 min at 4 °C. The soluble fraction was purified through Nickel magnetic beads with specificity to His-tag present in the protein’s N-terminal. The purity of OBP::CPPs was evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) under
44 reducing conditions. To remove the presence of salts and imidazole after purification the samples were dialyzed for 3 days, at 4 °C against ultrapure water. 2.5. Characterization of OBP::CPPs proteins 2.5.1. SDS-PAGE gel electrophoresis Lyophilized proteins were solubilized in 50 mM Tris-HCl pH 7.5, loaded on SDS-PAGE gel (12.5%) and stained with Coomassie solution to analyze size and purity. 2.5.2. MALDI-TOF mass spectrometry Mass/charge of OBP fusion proteins was verified by Matrix-Assisted Laser Desorption/Ionization with time-of-flight (MALDI-TOF) using sinapic acid (SA) as the matrix (≥ 99.5%). The mass spectra were acquired on an Ultra-flex MALDI-TOF mass spectrophotometer (Bruker Daltonics GmbH) equipped with a 337 nm nitrogen laser. A double layer deposition was used to analyze OBP fusion proteins. For this, a saturated solution of SA in ethanol, was deposited in the ground steel plate until dry. Each sample, previously dissolved in TA30 (30% acetonitrile/70% TFA), was mixed (1:1) with a saturated solution of SA in TA30. A volume of 2 μL of each mixture was spotted onto the ground steel target plate (Bruker part nº 209519) and analyzed using the reflective positive mode. 2.5.3. Circular dichroism spectroscopy The structural state of OBP::CPPs in the presence or absence of ligand (1-AMA) was investigated by circular dichroism (CD) spectroscopy, using a Jasco J-1500 spectropolarimeter equipped with a temperature controller. The structure of CPP peptides was also analyzed. CD spectra were recorded at 37 °C, using 10 μM as a fixed concentration for all the samples tested. The spectra were obtained over the wavelength interval of 180-260 nm at a scan speed of 20 nm/min and bandwidth of 1 nm. The path-length cell was 1 mm. Baseline was recorded with the same buffer of the samples (5 mM phosphate buffer, pH 7.5) and subtracted to the protein spectra. Final spectra were obtained by the average of three scans for each sample.
45 2.5.4. Fluorescence binding assays The binding capacity of wild-type OBP-I (OBP wt) and OBP::CPPs was determined by direct titration with 1-AMA, as reported in Silva et al. (2014).67 Briefly, the fluorescent probe 1-AMA was dissolved in 95% ethanol at 1 mM. Increasing ligand concentrations were added to 1 μM of each protein in 50 mM Tris-HCl, pH 7.5 and incubated at 37 °C for 1 h. Fluorescence emission spectra were recorded measuring the OBP-ligand complex formation by increase in the emission intensity at 481 nm when excited at 295 nm.67, 112 Measurements were recorded in triplicate, on a microplate spectrofluorometer (BioTek Synergy MX) equipped with a temperature controller. Dissociation constants (Kd) were calculated from a plot of fluorescence intensity versus concentration of ligand, obtained with a standard nonlinear regression method, described in Malpeli et al. (1998).113 2.6. Liposomes preparation Liposomes (18 mM) were prepared based in a formulation developed previously in our group, using 52.5% of 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 42.5% of cholesterol (CHOL) and 5.0% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPEPEG) through ethanol injection method.114 Further, the liposomes were extruded through 200 nm polycarbonate filters (Nucleopore) followed by several passages through 100 nm polycarbonate filters (Nucleopore) to reduce the size of the vesicles, using an extruder supplied by Lipex Biomembranes Inc.. The size distribution and surface charge of the liposomes were characterized by dynamic light scattering (DLS). 2.7. Dynamic Light Scattering The mean size diameter (nm), polydispersity index (PDI) and zeta-potential (mV) of the liposomes were measured in a Zetasizer Nano ZS (Malvern Instruments) at 25 °C. Prior to DLS measurements, the samples were diluted with PBS at pH 7.4 (for size) and with ultrapure water (for zeta-potential). All measurements were read in triplicate, being the results described as mean ± standard deviation. 2.8. Transduction of 1-AMA into liposomes reservoirs To determine the concentration of 1-AMA transduced to liposomes, a new procedure was designed and implemented (Figure 3.2). At step 1, 100 μM of OBP::CPPs were incubated with 100 μM of 1-
52 Table 3.1. Amino acid sequence, molecular weight and ionic charge of CPPs CPP Amino acid sequence Molecular weight (Da)[a] Total charge[a] Structure[b] Tat GRKKRRQRRRPPQ 1719.04 +8 pVEC LLIILRRRIRKQAHAHSK 2209.72 +6 Pep-1 KETWWETWWTEWSQPKKKRKV 2848.26 +3 [a] Values determined by Expasy ProtParam tool at physiological pH. [b] De novo peptide structure prediction in http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py#forms::PEPFOLD3. 3.1. Purity and size of fusion proteins The three OBP::CPP fusion proteins were characterized regarding purity and molecular weight. SDSPAGE gel confirmed the high purity of all the constructs purified using the nickel magnetic beads (Figure 3.4A). The migration pattern of the fusion OBPs do not correspond to the theoretical molecular weight of the proteins. OBP::pVEC (22.3 kDa) runs faster in SDS-PAGE than OBP::Tat (21.6 kDa). As other authors reported, proteins can in some cases display anomalous migration in SDS-PAGE relatively to protein standards. Other techniques such as mass spectrometry, like MALDI-TOF, are more appropriate to precisely measure the mass of the protein.137-138 Data obtained by MALDI-TOF confirmed the monodispersity of purified proteins whereas the experimental molecular weight is in accordance with the theoretical values (Figure 3.4B).
53 Figure 3.4. (A) SDS-PAGE electrophoresis of 100 μM of OBP wt, OBP::Pep-1, OBP::pVEC, OBP::Tat and Precision Plus ProteinTM molecular weight; (B) Theoretical (by SnapGene® 3.0.3) and quantified (by MALDI-TOF) mass of OBP::CPPs. 3.2. Structural analysis by CD spectroscopy The effect of CPPs on OBP structure was evaluated by circular dichroism (CD) spectroscopy. OBPI belongs to lipocalin superfamily that is known to share a conserved folding pattern, an eight stranded β-barrel flanked by a α-helix at the C-terminal end of the polypeptide chain.108 The shape of the spectra of OBP wt, OBP::Tat and OBP::Pep-1 is similar, i.e., the maximum and minimum, respectively, at 195 nm and 215 nm, are characteristic of a fold with a high content of β-sheets (Figure 3.5A). The fusion of OBP with Tat and Pep-1 did not disturb the protein conformation. The spectrum of OBP::pVEC (Figure 3.5A, dash line) displayed less pronounced peaks. This loss in intensity indicates a more extended state of the β-sheets resulting from the partial unfolding of OBP promoted by the pVEC peptide.139-140 The presence of 1-AMA did not significantly change the OBP::CPPs spectra as shown in Figure 3.5C. The differences detected are related with a protein rearrangement associated with the binding of the ligand to the aromatic residues of the OBP pocket.86, 141 CPPs alone showed a negative band at 195-205 nm characteristic of a random coil conformation (Figure 3.5B). This result is expectable for CPPs when in aqueous buffers. Nevertheless, these peptides can acquire other conformations in the presence of different solvents.112
54 Figure 3.5. Circular dichroism spectra of (A) OBP wt and OBP::CPPs; a close-up of pVEC is highlighted; (B) CPP peptides and (C) OBP::CPPs in the presence and absence of 1-AMA; for a better visualization of CD spectrum of CPPs and pVEC, different scales were used. 3.3. Binding capacity of fusion proteins The binding ability of OBP::CPPs was evaluated by fluorescence binding assay using 1-AMA as ligand model. When 1-AMA is at the binding site of OBP-I, the emission wavelength undergoes a minor blue shift and the intensity of the fluorescence is greatly increased.108 The free 1-AMA is monitored at 537 nm while the binding of 1-AMA to OBP is measured at 481 nm (λex = 295 nm). Binding strength,
55 measured in terms of the dissociation constant (Kd), revealed different behavior for the three OBP::CPPs (Figure 3.6). Binding curves obtained by fluorescence reveal similar dissociations constants for OBP::Pep-1 (Kd = 0.56 μM), OBP::Tat (Kd = 0.58 μM) and OBP wt (Kd = 0.44 μM) while a distinct behavior was observed for OBP::pVEC (Kd = 2.46 μM). The high dissociation value of OBP::pVEC fusion protein could be related with structure rearrangements induced by the presence of the CPP, as indicated by CD spectra in figure 3.5A. Figure 3.6. Binding curves obtained by measuring the fluorescence of 1 μM OBPs::CPPs in 50 mM Tris–HCl, pH 7.5, at equilibrium with several concentrations of 1-aminoanthracene. The dissociation constants were obtained at 37 °C by mathematical fitting of data.142 Values are the mean ± SD of 3 independent experiments. 3.4. Transduction of 1-AMA driven by OBP::CPPs into liposomes reservoirs A new methodology was developed to monitor 1-AMA transduction into liposomes membrane (Figure 3.2). Nontoxic and biodegradable143 liposomes were used as reservoirs of 1-AMA transduced by OBP::CPPs. To optimize the 1-AMA transduction efficiency, several incubation periods were tested (data not shown). We observed that 1 h of incubation at step 1 (OBP::CPPs + 1-AMA) and 1 h of incubation at step 2 (OBP::CPPs/1-AMA complex + liposomes) resulted in higher transduction
56 efficiencies. The results obtained (Table 3.2) revealed the role of OBP::CPP construct on the transduction efficiency of 1-AMA. OBP::Tat presented a transduction efficiency near 42%, whereas OBP::Pep-1 and OBP::pVEC, showed efficiencies of 31.6 and 17.2%, respectively. These differences can be attributed to the amino acid sequence of CPPs, their charge, and hydrophobic/hydrophilic character, and to the structural conformation of OBP.144 Peptides rich in arginine residues (R) like Tat, have been described as having higher transduction ability than CPPs rich in lysine residues (K), like Pep-1, and than peptides rich in leucine (L) and isoleucine (I) residues, like pVEC.145 According to the work of Herce et al. (2009), the arginine and lysine residues of Tat initially bind to the phosphate groups of phospholipids producing a strong alteration of the membrane. A pore is formed due to translocation of a single arginine residue to the phosphate group of DOPC.146 Recent work of Nischan et al. (2015) showed the transduction efficiency of cyclic Tat conjugated with green fluorescent protein (GFP) into the cytosol of living cells and compare it with an amphipathic peptide, PTD4.147 They observed that while Tat was successfully delivered into the cell, PTD4 was retained on the endosomes.147 Table 3.2. 1-AMA transduction efficiency driven by OBP::CPPs into liposomes and 1-AMA binding dissociation constants (Kd) of OBP::CPPs. The CPP effect was determined relatively to the OBP wt. Values are the mean ± SD of 2 independent experiments Protein Kd (μM) Efficiency (%) CPP effect (%) OBP::Tat 0.58 41.93 ± 0.73 16.50 OBP::Pep-1 0.56 31.64 ± 2.36 6.20 OBP wt 0.44 25.47 ± 0.16 - OBP::pVEC 2.46 17.27 ± 0.32 ≈ 0 Higher transduction efficiency of 1-AMA by OBP::Tat can also be associated to the high pKa of arginine which results on the delocalization of the positive charge of guanidinium side chain promoting the protonation of arginine even within membranes. This charge delocalization might contribute to the destabilization of the liposomes membrane.148-150 The hydrophilicity of CPPs plays also an important role on transduction efficiency of 1-AMA. According to Expasy ProtParam tool, the grand average of hydropathicity (GRAVY) indicated that the
57 three CPPs are hydrophilic, being Tat (-3.492) the more hydrophilic followed by Pep-1 (-2.038) and pVEC (-0.444).151 As more hydrophilic CPP is, higher levels of transduction are obtained.148 The lower transduction efficiency obtained by OBP::pVEC is associated to the destabilization of OBP structural conformation induced by the CPP, as confirmed by CD spectra (Figure 3.5A). The binding capacity of CPPs to 1-AMA was evaluated (data not shown) to confirm the role of OBP on the binding event. A residual fluorescence signal at 481 nm was detected indicating that 1-AMA is not binding to CPPs. 1-AMA in the presence of liposomes was performed as a control on the liposomal transduction experiments. This control revealed no 1-AMA transduction. Liposomes were physicochemical characterized by means of size, polydispersity index, and surface charge after transduction. Liposomes maintain the mean size with a small variation of the surface charge, being stable for at least 2 months of storage (Table 3.3). Table 3.3. Physicochemical characterization of liposomes before and after 1-AMA transduction Sample Time Size (nm) PDI Zeta (mV) Empty Liposome 0 days 104.6 ± 1.54 0.07 ± 0.02 -24.6 ± 1.13 2 months 107.6 ± 1.62 0.12 ± 0.04 -26.8 ± 0.64 Liposome + 1-AMA 0 days 102.7 ± 1.14 0.09 ± 0.02 -23.8 ± 1.18 2 months 106.3 ± 2.04 0.07 ± 0.02 -22.8 ± 1.05 Liposome + OBP wt + 1-AMA 0 days 105.1 ± 1.09 0.01 ± 0.02 -24.3 ± 0.68 2 months 106.7 ± 0.85 0.01 -22.9 ± 2.33 Liposome + OBP::Pep-1 + 1-AMA 0 days 104.1 ± 1.43 0.11 ± 0.03 -22.1 ± 1.48 2 months 106.5 ± 1.63 0.06 -23.9 ± 0.71 Liposome + OBP::pVEC + 1-AMA 0 days 103.7 ± 3.53 0.09 -25.3 ± 1.56 2 months 105.6 ± 0.15 0.08 ± 0.02 -23.6 ± 1.77 Liposome + OBP::Tat + 1-AMA 0 days 105.4 ± 1.66 0.08 ± 0.01 -24.7 ± 1.62 2 months 106.8 ± 0.78 0.12 ± 0.02 -22.8 ± 1.25 3.5. 1-AMA distribution evaluation by fluorescence spectrophotometry The prediction of 1-AMA distribution in the system was obtained by fluorescence spectra using the same conditions described previously (Figure 3.7). Maximum peaks were determined when 1-AMA is
58 free, bind to the protein and transduced into liposomes. The deconvolution of these peaks allowed to determine the percentage of 1-AMA distribution (Table 3.4) which was in accordance with the experimental transduction efficiency data obtained (Table 3.2). Figure 3.7. Fluorescence spectra of OBP wt, OBP::Pep-1, OBP::pVEC and OBP::Tat, obtained after 1 h of incubation at 37 °C (Ex. 295 nm).
59 Table 3.4. 1-AMA distribution after peak deconvolution 1-AMA distribution Experimental data from Table 3.2 Sample* [Free 1-AMA] (μM) [1-AMA complexed with OBP] (μM) [1-AMA] transduced through liposome (μM) [1-AMA] transduced through liposome (μM) OBP wt 48.5 51.5 - - OBP::Pep-1 59.0 41.0 - - OBP::pVEC 64.8 35.2 - - OBP::Tat 31.7 68.3 - - Liposome + OBP wt 41.4 33.0 25.5 25.5 Liposome + OBP::Pep-1 26.7 39.1 34.2 31.6 Liposome + OBP::pVEC 72.0 19.6 8.4 17.3 Liposome + OBP::Tat 12.9 46.4 40.7 41.9 *100 µM of 1-AMA was included in all samples. 3.6. Free energy calculations on 1-AMA transduction The potentials of mean force (PMF) calculations were performed to access the transduction of 1AMA by OBP wt. The free energy profile of 1-AMA transduced through the bilayer, obtained by umbrella sampling technique and WHAM analysis, is shown in Figure 3.8. The binding site of the protein corresponds to 0 nm (start point) in the z direction.
60 Figure 3.8. Free energy profile of 1-AMA in DOPE:DSPE:CHOL membrane and representative snapshots of 1-AMA moving in membrane normal axis, as background; water molecules and sodium ions were omitted for better visualization; 1-AMA molecule is shown in magenta spheres and OBP in green cartoon. For membrane, blue for nitrogen, red for oxygen, orange for phosphor and green for carbon. The PMF curve showed above indicates the probable distribution/behavior of 1-AMA at different regions of the system: complexed with the OBP, in the water layer, at the lipids head groups or in the interior of the membrane. According to the results obtained with PMF calculations, the most favored locations of 1-AMA are the internal cavity of OBP wt or inside the membrane (in the middle of the bilayer). We can observe the occurrence of a high energy barrier to unbind the 1-AMA from OBP, probably due to hydrogen bond loss at the complexed state, and from water layer to bilayer surface, as expected once the ligand is hydrophobic, showing repulsion when in these two polar environments. Qualitatively, we observed that 1-AMA encounter a cavity suitable for insertion while moving across the bilayer surface, and the membrane designed and equilibrated with our developed parameters, readjust without disorder to receive the ligand. The free energy profile indicates that 1-AMA can penetrate this model membrane when forming a complex with OBP wt. More important, we can predict that the closer the protein is to the membrane, promoted by the presence of CPPs the lower will be the energy barrier. The gap between protein and membrane is decreased by the presence of the CPPs.
61 4. Conclusions OBP intrinsic binding properties together with CPPs ability to penetrate into lipid membranes were explored for the capture and transduction of 1-AMA into liposome reservoirs. We reported for the first time the fusion of pig OBP with three cell penetrating peptides (Tat, pVEC and Pep-1). No structural changes of OBP were detected by circular dichroism spectroscopy after fusion with these peptides, except for pVEC. High binding affinity towards 1-AMA was observed for OBP::Pep-1 and OBP::Tat, whereas OBP::pVEC, due to the protein structural changes induced by the pVEC sequence, resulted in lower binding affinity. The transduction of 1-AMA driven by OBP::CPPs into liposomes is governed by the CPP amino acidic sequence, by their hydrophobic/hydrophilic character and by their charge. The highest 1-AMA transduction efficiency was obtained for OBP::Tat fusion protein. The new approach including OBP::CPP fusion proteins together with liposomes, as tailored reservoirs, open up new opportunities for the development of controlled systems to be used in a wide range of applications, namely for textiles functionalization, as biosensors, among others.
68 2.6 Functionalization of liposomes with OBP::SP-DS3 and OBP::GQ20::SP-DS3 Functionalized liposomes (18 mM) were prepared using 52.37% of DOPE, 42.37% of cholesterol, 5.00% of DSPE-PEG171 and 0.25% of engineered OBPs, through ethanol injection method. Firstly, the protein was added to a beaker containing Tris-HCl (50 mM), pH 7.5, preheated at 60 °C, under constant agitation (500 rpm), followed by the dropwise addition of the lipid content. To reduce their size, the OBP-functionalized liposomes were extruded using an extruder supplied by Lipex Biomembranes Inc., Vancouver, as previously described.36 The free protein, which was not anchored to the liposomes, was separated through the use of centrifugal filter tubes with a 100 kDa cut-off (Vivaspin 500). The efficiency of liposomes functionalization with the OBPs was assessed by SDS-PAGE and determined by the integration of the bands corresponding to the free and to the functionalized protein using the ImageJ software. For that, the initial amount of engineered OBPs added to the liposomes, the final protein-functionalized liposomes, and the non-anchored OBP were compared with specific bands of the molecular weight marker with a known amount of protein.173 The final data resulted from 10 software analysis and the efficiency was calculated as follows: Functionalization efficiency (%) = [initial protein] - [non-anchored protein] [initial Protein] ×100 where, functionalization efficiency corresponds to the amount (%) of protein anchored in liposomes; initial protein is the amount of protein added to the liposomal formulation; nonanchored protein is the free protein measured after liposomes functionalization. 2.7 Dynamic Light Scattering The ζ-potential (mV), mean size diameter (nm) and polydispersity index (PDI) of the functionalized liposomes (with and without 1-AMA) were evaluated using a Zetasizer Nano ZS (Malvern Instruments) at 25 °C. Measurements were recorded in triplicate, and the data was defined as mean ± standard deviation.
69 2.8 Transduction of 1-AMA into liposomes and binding affinity of 1-AMA to OBPs-functionalized liposomes To determine the 1-AMA transduction into liposomes and its capacity to bind OBPs functionalized in liposomes membrane, 1-AMA was added to the functionalized liposomes and incubated for 1 h at 37 °C, using 1 μM of the ligand and 1 μM of OBPs anchored to liposomes. After incubation the free 1AMA was removed by size-exclusion chromatography (SEC) using a 5 kDa cut-off PD-10 Desalting Column (GE-Healthcare). A control with nonfunctionalized liposomes was done using 52.5% of DOPE, 42.5% of cholesterol and 5.0% of DSPE-PEG, by ethanol injection method. The lipid content was resuspended in ethanol and added to a beaker containing Tris-HCl (50 mM), pH 7.5, pre-heated at 60 °C, with constant agitation (500 rpm). The size of the non-functionalized liposomes was reduced by extrusion, as described previously.36 Forty-five μM of 1-AMA was added to the liposomes and incubated for 1 h, at 37 °C. The 1-AMA transduced into the nonfunctionalized liposomes was separated from the free 1AMA using a PD-10 Desalting column (5 kDa cut-off). A control using the wild-type OBP was performed by adding the protein to the nonfunctionalized liposomes followed by the addition of 1-AMA. The liposomal mixture was incubated for 1 h, at 37 °C, and afterwards a 100 kDa membrane was used to separate the free wild-type OBP, the free 1-AMA and the OBP/AMA complex from the functionalized liposomes. The free 1-AMA was then separated from the fraction containing free OBP and OBP/AMA complex, using a PD-10 Desalting column (5 kDa cut-off). The fluorescence spectroscopy was applied to quantify the 1-AMA transduced into liposomes, the 1AMA bound to the OBPs and the free 1-AMA. The free 1-AMA was quantified by the fluorescence emission at 600 nm (excitation wavelength of 295 nm) and the concentrations were obtained using a fluorescence intensity versus 1-AMA concentration calibration curve. To determine the 1-AMA transduced into the liposomes, the fluorescence emission was recorded at 600 nm. The concentration of 1-AMA transduced was calculated using a calibration curve of 1-AMA and the 1-AMA bound to the OBPs was determined following the equation: [1−𝐴𝑀𝐴]𝑂𝐵𝑃 = [1 − 𝐴𝑀𝐴]𝑡𝑜𝑡𝑎𝑙 − ([1 −𝐴𝑀𝐴]𝑓𝑟𝑒𝑒 + [1 − 𝐴𝑀𝐴]𝑙𝑖𝑝𝑜𝑠𝑜𝑚𝑒𝑠)
70 where, [1-AMA]OBP corresponds to the concentration of 1-AMA bound to the protein, [1-AMA]total is the initial concentration of 1-AMA added to the liposomes, [1-AMA]free is the concentration of free ligand and [1-AMA]liposomes is the concentration of 1-AMA transduced into liposomes. Data were obtained in two independent experiments, and the results were presented as mean value ± standard deviation (SD). 3. Results and Discussion 3.1 Characterization of the engineered OBPs The purity and molecular weight of OBP proteins were characterized by SDS-PAGE gel and MALDITOF. The results confirmed the monodisperse character of the OBPs (Figure 4.2A), with the experimental molecular weight being in agreement with the theoretical values (Figure 4.2B). The wildtype OBP has 20054.64 Da, the OBP::SP-DS3 has 21357.16 Da whereas the OBP::GQ20::SP-DS3 has 25066.71 Da (Figure 4.2B). The binding of 1-AMA to the engineered OBPs was evaluated after 1 h, at 37 °C. The OBP::SP-DS3 (1.36 ± 0.16 μM) and OBP::GQ20::SP-DS3 (0.90 ± 0.11 μM) present higher dissociation constants (Kd) than wild-type OBP (0.44 ± 0.04 μM) (Figure 4.2C). These results point out the influence of SP-DS3 and GQ20 spacer on 1-AMA affinity to the engineered OBPs. Comparing both fusion proteins, there is an effect of the GQ20 spacer on the affinity of 1-AMA to OBP::GQ20::SPDS3 that can be attributed to differences on protein structures. CD studies of wild-type and fusion OBPs show a typical spectrum of proteins rich on β-sheets174 (Figure 4.2D), which is in accordance to the structure described for the wild-type OBP; a barrel composed by eight β-sheets with an internal cavity where the ligands are carried.4, 55, 76, 82 By CD spectrum analysis one can depict that OBP wt presents the β-sheets in a more extended state, when compared with the fusion proteins. The fusion of SP-DS3 and GQ20::SP-DS3 peptides with wild-type protein resulted in a differentiated decrease of the peak intensities at 195 nm and 215 nm, which might be associated with the α-helix content, or different orientations of the helix structure175, given by the presence of coil/unordered SP-DS3 peptide164 and the GQ20 spacer structures.175 The structural differences observed were also identified by predicting the protein structures of the three OBPs using I-TASSER (Iterative Threading ASSEmbly Refinement) online server (Figure 4.2E).176 The protein sequences were introduced and the structure models were
71 predicted based on matching known structures. From the models, we can identify the helix structure, predicted for SP-DS3, and the coil structure, expected for the GQ20 spacer. Figure 4.2. Characterization of engineered OBPs by SDS-PAGE gel electrophoresis (A); MALDI-TOF (B); binding dissociation constants (Kd) (C); CD spectroscopy (D) and I-TASSER structural models (E). In (A) is presented the run of purified wild-type OBP (1), OBP::SP-DS3 (2) and OBP::GQ20::SP-DS3 (3); M, Precision Plus ProteinTM Standards. In (B), theoretical mass including the His-tag and the m/z values are indicated; the wild-type OBP has 20054.64 Da, the OBP::SP-DS3 has 21357.16 Da whereas the OBP::GQ20::SP-DS3 has 25066.71 Da. In (C) the constants were determined after binding of the proteins with different concentrations of 1-AMA, 1 h at 37 °C. In (E) the arrows indicate the alterations added to wild-type protein. Competitive binding assays using two competitor fragrances, (R)-(-)-carvone 98% and (S)-(+)-carvone 96%, were performed to support the binding affinity of 1-AMA to the wild-type and to the two fusion proteins. The two selected fragrances are enantiomers, they are chiral pairs of leftand right-handed structures, containing an asymmetric carbon center. Mulla et al. (2015) reported a differentiated binding of (R)-(-)-carvone 98% and (S)-(+)-carvone 96% to mutated pig OBP (OBP-I F88W).76 Similarly,
72 our goal was to verify if both fragrances presented distinct binding behavior to OBP::GQ20::SP-DS3 and OBP::SP-DS3. The competitive assays of the fusion OBPs were measured using 2 μM of 1-AMA fluorescence probe, which is the maximum saturation value observed from dissociation curves (Figure 4.3A). The binding of the fragrance competitors to the proteins was monitored by the decrease of the fluorescence of the 1-AMA/OBPs complex (Figure 4.3B). Figure 4.3. Relative fluorescence versus 1-AMA concentration for OBP wt, OBP::SP-DS3 and OBP::GQ20::SP-DS3 when incubated at 37 ºC for 1 h (A). 1-AMA fluorescence competitive binding assay. Fluorescence emission spectra were recorded at 37 °C with 2 μM of 1-AMA in the presence of 1 μM of protein, pH 7.5 for 1 h, followed by addition of increasing concentrations of (R)-(-)-carvone 98% and (S)-(+)-carvone 96%, and incubated for 1 h at 37 °C (B). Excitation and emission wavelengths were 295 and 481 nm, respectively. Values represent the mean ± SD of 3 independent experiments. The dissociation constant values for fragrances range between 3.3 μM and 7.9 μM, showing a low binding by these competitors. The low binding competition of both fragrances enforces the high binding affinity of 1-AMA towards OBPs (Table 4.1). Furthermore, the decrease of the 1-AMA fluorescence intensity for both enantiomers has a different behavior, which supports the differentiated selectivity of the carvone enantiomers for the OBP proteins.
73 Table 4.1. Dissociations constant (KdF) for (R)-(-)-carvone 98% and (S)-(+)-carvone 96% fragrances determined by competitive binding Protein Fragrance KdF (μM) OBPwt (R)-(-)-carvone 3.29 ± 0.02 (S)-(+)-carvone 4.14 ± 0.02 OBP::SP-DS3 (R)-(-)-carvone 7.92 ± 0.003 (S)-(+)-carvone 5.94 ± 0.03 OBP::GQ20::SP-DS3 (R)-(-)-carvone 4.54 ± 0.10 (S)-(+)-carvone 6.43 ± 0.02 3.2. Liposomes functionalization with OBPs Liposomes were prepared by ethanol injection method at 60 °C, and vesicles with uniform and homogeneous size were obtained (Figure 4.4). For liposomes functionalization several protein concentrations were tested (data not shown). The maximal concentration achieved (≈ 45 μM), at which no protein precipitation was verified, corresponds to 0.25% of the liposomal content. Figure 4.4. Size distribution of liposomes functionalized with OBP::SP-DS3 (A) and with OBP::GQ20::SPDS3 (B) and for nonfunctionalized liposomes (C). (1) and (2) are independent measurements of replicates. To determine the level of OBP’s anchorage, we separated the OBP/liposome complex from the free protein through a 100 kDa membrane. The percentage of anchorage was evaluated by SDS-PAGE
74 electrophoresis (Figure 4.5). The analysis of the bands performed by ImageJ analyzer software (as described in section 2.6 of materials and methods), indicated that 97% of OBP::SP-DS3 and 92% of OBP::GQ20::SP-DS3 are anchored, revealing a high efficient functionalization of the liposomes. Figure 4.5. SDS-PAGE gel of liposomes functionalized with OBPs: (1) 45 μM OBP::SP-DS3; (2) liposomes with anchored OBP::SP-DS3; (3) nonanchored OBP::SP-DS3; (4) 45 μM OBP::GQ20::SP.DS3; (5) liposomes with anchored OBP::GQ20::SP-DS3; (6) nonanchored OBP::GQ20::SP-DS3 and (M) 4 μL Precision Plus ProteinTM Standards (BioRad). 3.3 Physicochemical characterization of protein-anchored liposomes The mean size distribution and the surface charge of the liposomes with anchored OBP::SP-DS3 and OBP::GQ20::SP-DS3 proteins were confirmed by dynamic light scattering measurements (Figure 4.4 and Figure 4.6). Both functionalized liposomal formulations exhibited particle sizes ranging between 110-116 nm and a monodisperse character (PDI near 0.10). By comparing both functionalized and nonfunctionalized liposomes, the latter presented smaller particle size (≈ 100 nm) and lower polydispersity (PDI ≈ 0.07), which might be associated with the functionalization of the liposomes with the engineered OBPs. The formulations remain stable until 180 days of storage at 4 °C, suggesting that the functionalization with OBP::SP-DS3 and OBP::GQ20::SP-DS3 proteins did not compromise the integrity of the liposomes.
75 Figure 4.6. Physicochemical characterization of nonfunctionalized liposomes (A, D) and liposomes containing the proteins OBP::SP-DS3 (B, E) and OBP::GQ20::SP-DS3 (C, F). Size, polydispersity index (PDI) and surface charge (ζ-potential) were measured using a Zetasizer Nano ZS. Values represent the mean ± SD of 3 independent experiments. Nogueira et al. (2015), have previously proved, through the measurement of tryptophan fluorescence, that SP-DS3 sequence inserts deeply into the membrane of liposomes.163 Here, we have also demonstrated that the OBP fused with the SP-DS3 peptide, with and without the GQ20 spacer, is inserted into the hydrophobic region of liposomes membrane, maintaining the bioactivity of the proteins (Figure 4.7). We do not have conclusive evidences about the construct’s localization (data not shown) because the tryptophan present in the OBP protein interfere with the fluorescence spectra at 280 nm of the SP-DS3 tryptophan inserted into the liposomal membrane.
76 3.4 Transduction of 1-AMA into liposomes and binding affinity of 1-AMA to OBPs-functionalized liposomes The transduction of 1-AMA into functionalized and nonfunctionalized liposomes and the binding affinity of 1-AMA to the OBPs was measured after 1h of incubation with 1-AMA, at 37 °C (Figure 4.7). The results indicate an effect of the SP-DS3 anchor peptide and of the GQ20 spacer in the transduction and binding of 1-AMA to liposomes functionalized with OBP::SP-DS3 and OBP::GQ20::SP-DS3. The presence of the GQ20 spacer in the OBP::GQ20::SP-DS3 protein increased the distance between the protein binding pocket and the liposomal surface, resulting in a reduction of the ligand transduction (18.6 ± 2.0%), when compared to the OBP::SP-DS3 protein (22.6 ± 1.3%). The proximity of the proteins to the lipid surface seems to influence the transduction of 1-AMA into liposomes, while the mobility imparted by the GQ20 spacer is essential for the 1-AMA binding. The binding efficiency of 1-AMA to the OBP::GQ20::SP-DS3 and to the OBP::SP-DS3 was 44.9 ± 3.9% and 29.0 ± 4.0%, respectively (Figure 4.7). The binding affinity is driven by the balance between the gain in binding energy and the loss of entropy related with conformational degrees of freedom.177 The length of the spacer is crucial in providing enough distance from the liposomes surface and accommodate the binding of ligand. As also reported by Fleiner et al. (2001), the best results of coupling efficiency were achieved when using longer PEG spacers.178 The presence of the spacer (increase of the distance and of the molecular mobility) affected differently the 1-AMA transduction efficiency into liposomes and the 1-AMA binding to the protein functionalized in liposomes. The ligand transduction efficiency was enhanced by the proximity (OBP::SP-DS3), while ligand binding efficiency was promoted by the molecular mobility (OBP::GQ20::SP-DS3). Wild-type OBP was used as a control of OBP::GQ20::SP-DS3 and OBP::SP-DS3 anchorage into liposomes. Because wild-type OBP does not have any anchor peptide, the amount of protein detected after separation probably resulted from adsorption at the surface of liposomes. The low percentage of binding (8.7 ± 0.3%) is associated with the low amount of OBP wt that remained adsorbed at liposomes surface after separation. On the other hand, the amount of 1-AMA on nonfunctionalized liposomes (14.2 ± 0.2%) might be associated with adsorption and permeation phenomena (Figure 4.7).
77 Figure 4.7. 1-AMA percentage transduced into liposomes and bound to the OBPs functionalized in liposomes after 1 h of incubation at 37 °C. (A) Experimental procedure to evaluate the amount of 1AMA transduced into the liposomes and bound to protein-functionalized liposomes. After incubation of liposomes with 1-AMA at 37 °C, the free ligand is removed using a gel filtration chromatography column with a 5 kDa cut-off. The amount of 1-AMA in the liposomes was measure by the fluorescence emission at 600 nm. (B) Experimental procedure to evaluate the amount of 1-AMA transduced into the liposomes and bound to wild-type OBP. The liposomes were incubated with protein and 1-AMA at 37 °C for 1 h. Then the free protein, free ligand and 1-AMA/OBP complex were separated from liposomes using a
84 Figure 5.2. Opposite temperature-dependent affinities of tOBP and OBP::GQ20::SP-DS3 to 1aminoanthracene (1-AMA). In (A) are presented the different binding affinities of tOBP and OBP::GQ20::SP-DS3. (B) is the schematic presentation of OBP’s competitive temperature-dependent mechanism; tOBP is presented in grey; OBP::GQ20::SP-DS3 is presented in magenta and 1-AMA is presented in green.
85 2. Materials and methods 2.1. Reagents Tris-base, imidazole, sodium phosphate, sodium chloride and dialysis tubes Midi 3500, capacity 50-800 μL, MWCO 3.5 kDa (Pur-A-Lyzer™ Midi Dialysis Kit) were available from MerckSigma, Spain. 1-aminoantrance (1-AMA) was purchased from TCI chemicals, Belgium. Nickel Magnetic Beads for His Tag Protein Purification was available from Biotool, Bimake, Spain. Molecular weight Precision Plus ProteinTM standards were purchased from BioRad, Portugal. All other reagents were acquired from MerckSigma and used as received. 2.2. Proteins production and purification Two proteins (tOBP and OBP::GQ20::SP-DS3) based on the sequence of OBP-I (PDB ID: 1DZK) were engineered. Truncated OBP (tOBP) resulted from the replacement of two phenylalanine residues at the binding pocket of OBP-I (F44A and F66A) and from the deletion of the first 16 residues of the Nterminal. OBP::GQ20::SP-DS3 is the result of the fusion of OBP-I with the anchor peptide SP-DS3 (DRDDQAAWFSQY) and a spacer of 20 repetitions of glycine-glutamine residues (GQ20). The OBP genes were synthetized by GenScript and cloned in pET-28a plasmid. OBPs were produced in Escherichia coli BL21(DE3) in Lysogeny broth (LB) medium with induction-cell at an optical density of 0.5-0.6 with Isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were harvest by centrifugation at 7,000 g, for 5 min at 4 °C, resuspended in phosphate buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4) supplemented with 10 mM of imidazole and lysed by sonication (40%, 3.0 sec ON, 9.0 sec OFF for 10 min) in sonicator vibracellTM SONICS. Soluble and insoluble fractions were separated by centrifugation at 12,000 g, for 30 min at 4 °C. The soluble fraction was purified through Nickel magnetic beads with specificity to His-tag present in the protein’s N-terminal. To remove the presence of high salts content and imidazole after purification the samples were dialyzed for 3 days, at 4 °C against ultrapure water. 2.3. MALDI-TOF mass spectrometry Mass of OBP proteins was verified by Matrix-Assisted Laser Desorption/Ionization with time-of-flight (MALDI-TOF) using sinapic acid (SA) as matrix (≥ 99.5%). The mass spectra were acquired on an Ultraflex MALDI-TOF mass spectrophotometer (Bruker Daltonics GmbH) equipped with a 337 nm nitrogen laser. A double layer deposition was used to analyze the OBPs. For this, a saturated solution of SA in
86 ethanol, was deposited in the ground steel plate until dry. Each sample, previously dissolved in TA30 (30% acetonitrile/70% TFA), was mixed (1:1) with a saturated solution of SA in TA30. A volume of 2 μL of each mixture was spotted onto the ground steel target plate (Bruker part n° 209519) and analyzed using the reflective positive mode. 2.4. Circular dichroism (CD) spectroscopy The structural state of OBPs was investigated by circular dichroism (CD) spectroscopy, using a Jasco J-1500 spectropolarimeter equipped with a temperature controller. Far-UV CD spectra were recorded in a 1-mm-path-length cell from 260 to 180 nm with a 1 nm resolution and at a scan speed of 20 nm/min. CD spectra were recorded at 25 and 37 ± 0.1 °C, using 10 μM as a fixed concentration. Baseline was recorded with the same buffer of the samples (5 mM phosphate buffer, pH 7.5) and subtracted to the protein spectra. Final spectra were generated by the average of three scans for each sample. 2.5. Fluorescence binding studies at 25 and 37 °C The ligand binding experiments were performed by direct titration with 1-AMA, as reported by Silva et al. (2014)67, at 25 and 37 °C. Briefly, the fluorescent probe 1-AMA was dissolved in 95% ethanol as 1 mM stock solutions. Successive increasing ligand concentrations (in buffer solution) were added to 1 μM of proteins and incubated at 25 and 37 ± 0.1 °C, for 15 min, in a microplate spectrofluorometer (BioTek Synergy MX) equipped with a temperature controller, with slits set at 5 nm bandwidth. The fluorescence emission spectra were recorded in three independent experiences and read in triplicate, measuring the OBP-ligand complex formation by the increase of emission intensity at 481 nm when excited at 295 nm.67, 112 Dissociation constants (kd) were calculated from a plot of fluorescence intensity versus concentration of ligand, obtained with a standard non-linear regression method, described by Malpeli et al. (1998).142 2.6. Temperature competitive-binding of OBPs to 1-AMA To study the competition between tOBP and OBP::GQ20::SP-DS3 for 1-AMA we developed a system constituted by the two OBPs separated by a dialysis membrane with a cut-off only permeable to 1-AMA (3.5 kDa). tOBP was placed inside the dialysis tube and OBP::GQ20::SP-DS3 was placed outside, in a
87 beaker. Initially, 1 mM of 1-AMA was added to the dialysis tube and incubated at 25 °C until equimolar equilibrium (172 μM). Further, 172 μM of the tOBP was added inside of the dialysis tube and 172 μM of OBP::GQ20::SP-DS3 was added in the beaker. After incubation at 25 °C for 24 h the concentration of 1-AMA in both compartments was measured by fluorescence spectroscopy. Afterwards the temperature of the system was increased until 37 °C and maintained during 24 h. After this period 1AMA concentration was measured in both compartments. The temperature of the system was lowered to 25 °C, and the concentration of 1-AMA was again measured after 24 h of incubation. The concentration of 1-AMA was determined by measuring the fluorescence of free 1-AMA fractions in the dialysis tube and in the beaker at 600 nm (λex = 295 nm) and replacing the experimental value in the calibration curve of fluorescence versus 1-AMA concentration. All steps were visually evaluated by photographic record. Measurements were recorded in two independent experiments and the results were expressed as mean value ± standard deviation (SD). 2.7. Statistical analysis The values reported in the circular dichroism spectra were generated by the average of three scans for each sample. The CD data were fitted with a Boltzmann sigmoidal line shapes. 2.8. Molecular Dynamics Simulations tOBP and OBP::GQ20::SP-DS3 were designed with PyMOL117, based on the OBP experimental structure 1DZK191, from Protein Data Bank (PDB)115 and using an equilibrated wild-type OBP. For our previous work on wild-type OBP171, we simulated OBP-I for 60 ns. We took a representative structure from these simulations to proceed with the necessary changes to the construction of the OBP-based proteins proposed here. Both proteins were modeled in water with the simple point charge (SPC) water model in an octahedral box with a hydration layer of at least 1.5 nm between the peptide and the walls. Na+ ions were added to neutralize the simulation boxes. One stage of energy minimization was performed using a maximum of 50,000 steps with steepest descent algorithm for both structures. The systems were initialized in a NVT ensemble, using V-rescale192 algorithm, with the coupling constant τT = 0.10 ps, to control temperature at 298K (25 °C) and 310K (37 °C), i.e. each system was settled to generate two independent runs at each temperature. After that, a NPT initialization step was performed, with V-rescale and Parrinello-Rahman barostat121 algorithms to couple temperature and
88 pressure at 298K/310K and 1 atm respectively. We used the following coupling constants: τT = 0.10 ps and τP = 2.0 ps. Position restraints (with force constant of 1000 kJ·mol-1·nm-2) were applied to all protein heavy atoms in initialization. 20 ns of MD simulations were performed for each system, without position restraints, and with the same NPT ensemble described above. All simulations were performed using the GROMACS 4.5.4 version123-124, within the GROMOS 54a7 force field (FF)125. The Lennard-Jones interactions were truncated at 1.4 nm and using particle-mesh Ewald (PME)122 method for electrostatic interactions, also with a 1.4 nm cut-off. The algorithm LINCS118119 was used to constrain the chemical bonds of the peptides and the SETTLE120 algorithm was used in the case of water. 2.9. MD simulations analysis MD simulations were performed to equilibrate the two engineered proteins at 25 and 37 °C. From MD simulations in water, at 25 and 37 °C, we computed the central structure (CS) of each engineered protein, for the last 15 ns of simulation time. These conformations minimize the RMSD variance when fitted against all other conformations of the trajectory, corresponding to the most populated conformation of the simulation. For these systems, we fitted the backbone and calculated the backbone RMSD (Root Mean Square Deviation). Then from the RMSD matrix, we extracted the most representative conformation of each simulation. The Secondary Structure (SS) profile was also computed by the Dictionary of Secondary Structure in Proteins (DSSP) method, from Kabsch and Sander (1983)193 that allows to calculate the percentage of each SS founded along the simulation time, using the hydrogen bond pattern. Both tools are implemented on GROMACS. The average binding pocket size was determined measuring the distance between the center of mass of tyrosine 52 (Tyr52) and isoleucine 100 (Ile100), over time. These amino acids are in opposite directions and symmetrically arranged in the β-barrel for all protein models. 2.10. Molecular Docking and MD Simulations on 1-AMA/OBPs complexes Docking experiments were performed using AutoDock Vina194 and prepared with the AutoDock Tools Software.127 We used the Central Structures at each temperature, obtained from the first round of MD simulations for the docking experiments. AutoDock Vina requires a grid spacing of 1 Å, generating boxes with approximately 28 x 30 x 30 grid points, for all systems. It uses a combination of scoring
89 function and an optimization algorithm, being fastest in predict poses. We used an exhaustiveness of 15, num_modes = 50 and energy range = 3. The amino acid residues of the proteins that interact with the 1-AMA were identified through the AutoDock tools at 25 and 37 °C. However, as the binding energies differ only in ≈ 0.5-1.0 kcal/mol between the systems, at both temperatures, we look at docking results only to see the interaction binding mode and extract the best binding pose to proceed to MD simulations of the complexes. 10 ns of MD simulations were performed for the 4 complexes obtained in the previous step. The same protocol applied to the free proteins simulations was used to simulate the complexes. The stability of each complex was followed through directly visualization of each trajectory. We also generate a central structure from these simulations, to serve as the most representative complex conformation. From the 10 ns MD simulations, we performed MM_PBSA calculations (Molecular Mechanics Poisson-Boltzmann Surface Area) to estimate the binding energy of the four AMA/OBPs complexes along time, using g_mmpbsa tool.195 We set the temperature for these calculations according to the simulations temperature, i.e. 25 or 37 °C. 3. Results and discussion 3.1. OBPs characterization The engineered OBPs were characterized regarding purity and molecular weight by Matrix-Assisted Laser Desorption/Ionization with time-of-flight (MALDI-TOF). The data obtained by MALDI-TOF confirmed the monodisperse character of the proteins with the experimental molecular weight (17832.53 kDa for tOBP and 25065.29 kDa for OBP::GQ20::SP-DS3) in accordance with the theoretical values (Figure 5.3). The SDS-PAGE gels (Figure 5.3) also confirmed the purity of OBPs.
90 Figure 5.3. MALDI-TOF and SDS-PAGE gel of tOBP (A) and OBP::GQ20::SP-DS3 (B). Theoretical mass and the m/z values are indicated in (C). In (D) is shown the original gel from that was grouping the gels indicated in (A) and (B), being the truncated OBP the run of the line 2 and the OBP::GQ20::SP-DS3 the run of the line 4. 3.2. Structural analysis Dynamic changes in OBPs secondary structure triggered by temperature alterations was verified by circular dichroism (CD) spectroscopy and by molecular dynamic simulations (MD) at 25 and 37 °C. The CD spectra of the tOBP and the OBP::GQ20::SP-DS3 revealed the maximum and the minimum peaks around of 195 nm and 215 nm, respectively (Figure 5.4 and figure 5.5A, D). This spectrum shape is characteristic of a fold with a high content of β-sheets, which is in accordance with mammalian OBPs structure known to share a conserved folding pattern: an eight stranded β-barrel flanked by an α-helix at the C-terminal end of the polypeptide chain.108, 189 The CD spectra of both OBP variants confirmed the effect of the mutations on the protein structure comparing with the wild-type spectra (Figure 5.4). We observed an alteration of the tOBP and OBP::GQ20::SP-DS3 conformations at 25 and 37 °C, which was more evident for the tOBP. The less pronounced peaks displayed by this protein might be attributed to a more extended state of the β-sheets resulting from the partial unfolding of
91 tOBP promoted by the deletion of the first 16 residues on the N-terminal. The fusion of the GQ20::SPDS3 sequence with the wild-type OBP resulted in an increase of the helix content given by the presence of the coil/unordered SP-DS3 peptide164 and of the GQ20 spacer structures175, 196, as evidenced in the spectra (Figure 5.4). Figure 5.4. Circular dichroism (CD) spectra of wild-type OBP, truncated OBP (tOBP) and OBP::GQ20::SPDS3, at 25 and 37 °C. Final spectra were generated by the average of three scans for each sample. The engineered OBPs present different spectra related with some structural differences which could affect the binding pocket conformation and affinity towards molecules. Comparing both CD spectra of tOBP at 25 and 37 °C, is evident a difference around 195 nm (Figure 5.5A). At 25 °C, tOBP adopts a barrel structure in which the hydrogen bonds network becomes looser, promoting the opening of tOBP pocket cavity.108 The CD spectra of OBP::GQ20::SP-DS3 at 25 and 37 °C show differences in the region of 215 nm (Figure 5.5D). This variation can be associated to the increase of helix content from the SPDS3 and GQ20 spacer.196 From MD simulations, we took the central structure (CS), for each designed OBP, at each temperature, to compare the structural features among them. CS represents the most probable conformation for each protein under the simulation conditions. The convergence of simulated systems was traced by Root Mean Square Deviation (RMSD) analysis and compared with the previously simulated for wild-type OBP171. The wild-type form is very stable and conserved as we can see by RMSD, in Figure 5.6. Figure 5.6 presents the RMSD plots for wild-type OBP, tOBP and OBP::GQ20::SP-DS3. RMSD for the new OBPs remains within an acceptable range and similar to the observed for wild-type
92 OBP. RMSD of the engineered OBPs are in agreement with the experimental results, tOBP is more stable at 25 °C and OBP::GQ20::SP-DS3 at 37 °C. Figure 5.5B-C corroborates the CD spectra results, i.e., when superimposed, the tOBP structures, demonstrated a more loose structure at 25 °C (grey structure) and a smaller barrel at 37 °C (cyan structure). OBP::GQ20::SP-DS3 displayed an opposite behavior (Figure 5.5E-F), presenting a larger barrel at 37 °C (magenta structure), whereas at 25 °C a smaller barrel is observed (grey structure).
93 Figure 5.5. Structure of engineered OBP proteins analyzed by circular dichroism spectroscopy and molecular dynamics simulations. Secondary structure spectra determined by circular dichroism (CD) spectroscopy of tOBP (A) and OBP::GQ20::SP-DS3 (D); Top (B) and side (C) views of tOBP superimposed central structure; Top (E) and side (F) views of OBP::GQ20::SP-DS3 superimposed central structure. For all images, grey represents the structures at 25 °C, cyan and magenta represent the structures at 37 °C for tOBP and OBP::GQ20::SP-DS3, respectively. Percentage of secondary
100 (position and interactions). The interactions between 1-AMA and the proteins, observed for the best docked pose (more negative ∆G value), are shown in figure 5.10. These docking positions were then submitted to 10 ns of MD simulation to follow the stability of each complex. Figure 5.10. Interaction bind mode of 1-AMA to OBPs estimated through molecular docking with AutoDock Vina. 1-AMA interaction with tOBP at 25 °C (A) and at 37 °C (B). Ligand bind mode of 1AMA to OBP::GQ20::SP-DS3 at 25 °C (C) and at 37 °C (D). 1-AMA ligand is presented in blue spheres and the amino acids involved in hydrogen bonds or van der Waals contacts, in CPK sticks representation. The molecular modelling simulations revealed that OBPs undergo slight structural changes that may lead to the binding of 1-AMA to other sites rather than the preferential position. Looking at 1-AMA positions, we see that the ligand does not bind to the same region in all cases, for the best docked poses (similar poses were seen in all systems but ranked with less favored ∆G). PyMOL can display
101 the cavities and pockets within the interior of a given molecule and looks at the complemental geometry and interactions between the ligand model and the protein. Using this tool we highlight the cavities that potentially can accommodate the 1-AMA ligand (Figure 5.10). The docking results are in accordance with the PyMOL predictions and the experimental binding assays. 1-AMA binds preferentially to tOBP at 25 °C (Figure 5.11A left) while to OBP::GQ20::SP-DS3, 1-AMA binds preferentially at 37 °C (Figure 5.11B right), supported by the highest number of PyMOL docking possibilities. Molecular docking has also been used as useful tool for analyzing the ligand interactions with the protein structure.90 Thereby, using the AutoDock tools, we identified the amino acid residues of both OBPs which interact with 1-AMA, at 25 and 37 °C (Table 5.1). The main interactions occur with nonpolar (hydrophobic) and polar amino acids. Some of these amino acids were previously described as residues of wild-type OBP-I interacting with odorants. Vincent et al. (2000) elaborated a list of residues of the OBP-I cavity which interact with different odors. Asn102 residue was described as interacting with three aromatic ligands: benzophenone (BZP), benzyl-benzoate (BZB) and 2-iso-butyl-3metoxypyrazine (IBMP).191 Also, Met114 was recognized as being involved in the interaction between OBP-I and BZB191. Both of these residues were also identified in our study for OBP::GQ20::SP-DS3 at 37 °C. In another study, molecular simulation analysis identified the Tyr82 residue and nearby residues as forming the cavity entry. Tyr82 is in fact a residue conserved in many OBPs.90 Additionally, the energetic analysis showed a high van der Waals interaction between the OBP and the odorants, representing more than 80% of the interaction energy compared with no strong hydrogen bond.90 In the molecular dynamic study of Golebiowski et al. (2006), the Asn86 and Asn102 residues of OBP-I were identified as being involved in the binding of the 2,6-dimethyl-7-octen-2-ol (DHM).90 Nagnan-Le Meillour et al. (2009) performed molecular studies from crystallized pig OBP complexed with undecanal (UND). The results showed that Tyr82 and Phe35 residues participate in the binding process. Phe55 was also identified but in a less extent.84 All of these residues are located at the border of the binding pocket and were identified in our study. Meillour et al. (2009) confirmed the simulation results by performing mutations in Tyr82 and Phe35. Fluorescence spectroscopy results indicated that both residues are involved in the binding of 1-AMA since the singles and double mutants were unable to bind 1-AMA.84 The study revealed that the Phe38 residue is also involved in the binding to UND. Some charged residues (Lys28, Asp110) participate on the dissociation via their side chain hydrogen atoms84. These amino acids, Lys28 and Asp110, were also identified in our simulation analysis. Our study is a step
102 further the existing knowledge since we describe other residues involved in the binding of 1-AMA to OBPs (Table 5.1, in bold). Table 5.1. Amino acid residues of tOBP (tOBP) and OBP::GQ20::SP-DS3 involved in the 1-AMA binding. The analysis was performed using the AutoDock Vina at 25 and 37 °C. In bold are described the new residues involved in the binding of 1-AMA to OBPs yet not identified in literature tOBP OBP::GQ20::SP-DS3 25 °C 37 °C 25 °C 37 °C Hydrogen Bond VAL105 ILE29 None ASP110 van der Waals PHE35/ ALA83/ ASN104 PHE38/ VAL147/ ILE149 ASN32/PRO34/ PHE55/ LYS58/ TYR82/ ARG152 LYS28/ PHE35/ ASN86/ASN102/ ASN104/VAL105/ ASP106/MET114 From 10 ns MD simulations on docking poses, we observed that the tOBP at 37 °C and OBP::GQ20::SP-DS3 at 25 and 37 °C, conserved the docked pose and the interactions with 1-AMA. Interestingly, for tOBP at 25 °C was verified a spontaneous movement of the 1-AMA to the interior of the β-barrel. This result is in great agreement with experimental data, which consider this protein as the ablest to carry the ligand at this temperature (Kd = 0.45 μM at 25 °C). Figure 5.11 shows a superposition of docked and simulated positions for 1-AMA, for the two proteins at both temperatures.
103 Figure 5.11. Comparison between docked position and middle structure from MD simulations. The position of 1-AMA was estimated through molecular docking (1-AMA in cyan spheres) and through MD simulation (1-AMA in green spheres). A and B display the cavities estimated with PyMOL, in blue surface, for tOBP at 25 and 37 °C (A) and OBP::GQ20::SP-DS3, at 25 and 37 °C (B). In C is presented the most probable position for 1-AMA interaction with tOBP, at 25 and 37 °C, respectively. In D is shown the most probable bind mode of 1-AMA to OBP::GQ20::SP-DS3, at 25 and 37 °C, respectively. We see in Figure 5.11C-D that MD simulation generates a middle structure around the docked position. In these cases, 1-AMA moves in the same binding region sampling the docked position along the simulation, although with a central structure, derived from the simulation in a slightly different position. The stability of the complex was then proved, using MD simulations, in these three cases: OBP::GQ20::SP-DS3/1-AMA, at 25 and 37 °C, and tOBP/1-AMA at 37 °C. The interactions with the same group of amino acids observed by docking (Table 5.1) are sampled along the simulation time. In the case of 1-AMA sampled in complex with tOBP at 25 ºC, the ligand demonstrates a spontaneous
104 movement toward the interior of the β-barrel. This fact might be an indication that under dynamics, the system is able to rearrange to better accommodate 1-AMA, protecting this hydrophobic ligand from the water environment. To give an estimation about the free energy vs time interaction, we use the g_mmpbsa tool195 to follow the binding energy along the simulated complexes trajectories. Figure 5.12 shows the ligandprotein binding energy, for both systems at both temperatures, supporting the preference of 1-AMA for tOBP at 25 °C and for OBP::GQ20::SP-DS3 at 37 °C. These results are presented as running average curves in Figure 5.12. Figure 5.12. Binding energy along time obtained from g_mmpbsa tool. Running average binding curves in kJ/mol, of 1-AMA/tOBP complexes in grey and cyan, at 25 and 37 °C respectively and for 1AMA::OBP::GQ20/SP-DS3 complexes in black and magenta, at 25 and 37 °C respectively. 4. Conclusions We have constructed two new OBPs based on the sequence of OBP-I aiming different goals. tOBP was engineered to impart selective porosity while OBP::GQ20::SP-DS3 was designed to anchor to lipid membranes. Our data on the ability to bind 1-AMA revealed that tOBP and OBP::GQ20::SP-DS3 had opposite binding affinities depending on the temperature. Based on this finding we have explored the thermo-responsive behavior of the engineered OBPs to study the movement of 1-AMA between them
105 using temperature as trigger. We have observed that the binding affinity of the two OBPs was directly related with their structural conformation induced by temperature. The dissociation constants confirmed the high affinity of 1-AMA to tOBP at 25 °C (Kd = 0.45 μM) and to OBP::GQ20::SP-DS3 at 37 °C (Kd = 0.58 μM). The binding pocket size and the bend content were considered essential to understand the thermo-responsive behavior of the OBPs. Both parameters were evaluated by CD spectroscopy and MD simulations confirming their fundamental contribution to OBPs behavior. The average size of the pocket is directly related with OBPs’ binding affinity while the bend content is inversely proportional. At 25 °C tOBP presents a wider pocket size and a more relaxed structure than OBP::GQ20::SP-DS3, thus showing higher affinity to 1-AMA. At 37 °C, we observed a higher bend content indicating conformational alterations of tOBP resulting in a smaller pocket size, therefore decreasing the binding of 1-AMA. At this temperature, OBP::GQ20::SP-DS3 presented an opposite behavior, lower bend content and larger pocket size, resulting in an higher binding of 1-AMA compared with tOBP. We found a temperature-dependent affinity competition between the two OBPs when placed in the same system. When triggered by temperature there was a reversible displacement and movement of 1-AMA from one OBP to the other. Docking experiments also characterized the 1-AMA binding preference and locations of the ligand in the two engineered proteins. The MD data are in agreement with the experimental results. The 10 ns of MD simulation confirmed the docking results for tOBP/1AMA at 37 °C and OBP::GQ20::SP-DS3/1-AMA, at 25 and 37 °C. 1-AMA binding to tOBP at 25 °C shows a spontaneous phenomenon of insertion in the β-barrel. The modeling techniques have shown variations on the binding site, resulting from the structural and dynamic profile conferred by temperature changes. This suggests that OBP-based proteins are able to adjust and offer new binding locations at the entrance of the pocket, while keeping their carrier function. In summary, the engineered OBPs explored in this work showed tunable affinities upon temperature changes. This feature was described for the first time for this class of proteins opening space to the exploitation of a new class of functional materials.
106 Chapter 6 Release of Fragrances from Cotton Functionalized with Carbohydrate-Binding Module Proteins
107 Release of Fragrances from Cotton Functionalized with Carbohydrate-Binding Module Proteins Abstract Perspiration as response to daily activity and physical exercise results in unpleasant odors that cause social unrest and embarrassment. To tackle it, functional textiles incorporating fragrances could be an effective clothing deodorizing product. This work presents two strategies for the release of βcitronellol from functionalized cotton with carbohydrate-binding module (CBM)-based complexes (OBP::GQ20::CBM/β-citronellol – approach 1 and CBM::GQ20::SP-DS3-liposome/β-citronellol – approach 2). CBM from Cellulomonas fimi was fused with the odorant-binding protein (OBP::GQ20::CBM) and with an anchor peptide with affinity to liposomes membrane (CBM::GQ20::SPDS3). In approach 1, OBP fusion protein served as fragrance container, whereas in approach 2, the fragrance was loaded into liposomes with a higher cargo capacity. The two strategies showed a differentiated β-citronellol release profile triggered by an acidic sweat solution. OBP::GQ20::CBM complex revealed a fast release (31.9% and 25.8% of the initial amount, after 1.5 h and 24 h of exposure with acidic sweat solution, respectively) while the CBM::GQ20::SP-DS3-liposome complex demonstrated a slower and controlled release (5.9% and 10.5% of the initial amount, after 1.5 h and 24 h of exposure with acidic sweat solution, respectively). Both strategies revealed high potential for textiles functionalization aiming at controlled release of fragrances. The OBP::GQ20::CBM/β-citronellol complex is ideal for applications requiring fast release of a high amount of fragrance, whereas the CBM::GQ20::SP-DS3-liposome/β-citronellol complex is more suitable for prolonged and controlled release of a lower amount of β-citronellol.
108 1. Introduction In response to unpleasant odors resultant from daily activity and physical exercise, there is an increasing need for safe and effective clothing deodorizing products.198-199 Smart textiles have arisen as new textiles that can incorporate functional elements added in finishing of textiles and may respond to changes like light, temperature, mechanical stress or humidity.200 The use of fragrances is often essential to create an elegant and welcoming environment, particularly in daily social interplay.201 This way, the encapsulation and the release of fragrances from functionalized fabrics has arisen as a great strategy for the development of stimulus responsive cosmetotextiles. Abdelkader et al. (2018) described the preparation of nanocapsules containing 2-ethoxynaphthalene (neroline) fragrance by interfacial polycondensation method for cotton functionalization.202 Hu et al.203 produced polybutylcyanoacrylate (PBCA) nanocapsules encapsulating rose fragrance and evaluated its release from cotton fabrics by gas chromatography–mass spectrometry (GC-MS).203 Some studies showed that the complexation of β-cyclodextrins with fragrances allow retention of fragrances for a long period of time.204 An example of cyclodextrins application in textile processing is on the entrapment of aroma from sweats and cigarette smoke. Despite all the strategies already developed and implemented for fragrance release in textiles, other approaches have emerged for the functionalization of textile surfaces, namely based on stimulus-responsive materials. Carbohydrate-binding module (CBMs), previously named cellulose-binding domains (CBD), are noncatalytic modules of enzymes promoting the association of the enzyme to the substrate.205-207 These modules have been used for the functionalization of fibers in paper and textile industries. For example, the CBMN1 from Cellumonas fimi cellulase has the capacity to be adsorbed on cotton at pH of around 7 for long periods of time without damaging cellulose.208 Cadena et al. (2010) reported the use of recombinant CBM3b, originally from Paenibacillus barcinonensis endoglucanase Cel9B, to alter the cellulose fiber surface and thus to improve the paper properties.209 A patented product composed of fragrance-bearing particles conjugated to CBMs was added to laundry powder, thereby reducing the amount of fragrance needed in the product.210 In recent decades, several works have reported the encapsulation of active compounds into liposomes for health and cosmetics applications.211,212,213 The liposomes have the capacity to entrap different kinds of molecules and can be functionalized for a specific target. In our previous work, we explored the application of liposomes functionalized with OBP-I as nanodevices for odorant molecules
109 retention useful for textile and cosmetic applications.214 In nature, odorant binding proteins (OBPs) have the capacity to bind and release fragrances.33 Silva et al. (2014) explored, for the first time, the use of pig OBP-I to functionalize cotton fabrics, by electrostatic affinity, for the release of fragrances and the reduction of unpleasant odors, like cigarette smoke.67 In this work, we explored two strategies for the release of β-citronellol from cotton functionalized with CBM-based fusion proteins. In the first strategy, pig OBP-I from Sus scrofa was fused with the CBMN1 from Cellulomonas fimi (OBP::GQ20::CBM) and incubated with the fragrance prior to cotton functionalization. In the second strategy, the CBMN1 was fused with SP-DS3 peptide (CBM::GQ20::SPDS3) to anchor the protein in the liposomes containing the fragrance. The CBM::GQ20::SP-DS3liposome complex was further applied on cotton. Both fusion proteins were designed by including a spacer (GQ20) to confer conformational mobility to the fused partners.152, 157, 214 For both strategies, the release profile was evaluated by GC-MS considering the response to an external stimulus (acidic sweat solution; pH = 4.3 ± 0.2, as indicated in AATCC method 15-2009 “Colorfastness to Perspiration”). The release of β-citronellol triggered by acidic sweat solution mimicks the conditions of perspiration relying on the dissociation of the fragrance from OBP and on the release from liposomes. The two approaches here presented were designed to develop new cosmetotextiles for the release of fragrances. This technological solution can stimulate the textile industry in the search for new solutions creating a set of new perspiration-related products. 2. Materials and Methods 2.1. Reagents Tris-base, imidazole, sodium phosphate, sodium chloride, cholesterol and SPME fiber (100 µm polydimethylsiloxane) were acquired from MerckSigma, Spain. Nickel Magnetic Beads for His Tag Protein Purification was available from Biotool, Bimake, Spain. GRS Protein Marker Blue and GRS Unstained Protein Marker were purchased from GRISP, Portugal, as well as the culture medium. 1aminoanthracene (96%), β-citronellol (92%), coumarin (99%), vanillin (98%) and eugenol (99%) were acquired from TCI chemicals, Belgium. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) were purchased from Lipoid, Canada. All other reagents were acquired from MerckSigma, Spain and
116 operated in electron ionization (EI) mode at 70 eV with total ion chromatogram (TIC) detection mode for quantitative determination and S/N ratio of 5. Calibration curves of β-citronellol were determined using the same conditions of the samples (temperature and time). Each time-point was evaluated separately, and all the measurements were done in duplicate. The amount of β-citronellol was determined by integration of the peaks from chromatograms and quantified against the calibration curves. 3. Results and discussion 3.1. Strategies for β-citronellol release from functionalized cotton fabrics Two strategies for β-citronellol release from cotton functionalized with CBM-based complexes, OBP::GQ20::CBM and CBM::GQ20::SP-DS3-liposome, are proposed here. In the first strategy, OBP::GQ20::CBM protein (20 μM) is incubated with 40 μM β-citronellol to promote the binding of the fragrance to the protein. The OBP::GQ20::CBM/β-citronellol complex is then applied for cotton functionalization. The release of the fragrance from OBP is triggered by an acidic sweat solution and measured by GC-MS chromatography. In the second strategy, liposomes with CBM::GQ20::SP-DS3 anchored in the membrane and encapsulating 1000 μM of β-citronellol are used for cotton functionalization. As for the strategy 1, the release of β-citronellol from liposomes is herein triggered by addition of an acidic sweat solution and evaluated by GC-MS (Figure 6.1).
117 Figure 6.1. Schematic presentation of β-citronellol release from functionalized fabrics. The selection of the fragrance was made based on the binding properties of OBP. The binding affinity of OBP::GQ20::CBM was evaluated using 1-AMA as model ligand, at pH 7.5. The ligand revealed a high affinity to the protein at this pH value (Ka = 2.56 ± 0.04 μM) (Table 6.1). The binding of several fragrances to the OBP was determined by competitive assays to select the molecule with the best affinity towards the protein. These studies indicate that β-citronellol is the fragrance with the highest affinity towards OBP (Table 6.1). Table 6.1. Association constants (Ka) of OBP::GQ20::CBM for 1-AMA (ligand model) and four fragrances (β-citronellol, coumarin, vanillin and eugenol) at 37 °C, at pH 7.5 for 1 h. Association constants (Ka) (μM-1) 1-AMA β-citronellol Coumarin Vanillin Eugenol 2.56 ± 0.04 4.17 ± 0.05 0.21 ± 0.02 0.18 ± 0.01 0.31 ± 0.04
118 The amount of CBM::GQ20::SP-DS3 protein anchored in the liposomal membrane was assessed by SDS-PAGE gel electrophoresis (Figure 6.2A). The amount of anchored protein was estimated using the ImageJ analysis, by comparison of liposomal formulation band intensity with the specific bands of the molecular weight marker, as reported in Gonçalves et al. (2018).214 The analysis inferred that about 99% of CBM::GQ20::SP-DS3 is anchored in the membrane of liposomes. The nonencapsulated β-citronellol was separated from liposomes through a membrane with 100 kDa cut-off and quantified using GC-MS. The GC-MS spectrum for quantification of nonencapsulated β-citronellol does not present any peak related with β-citronellol (Figure 6.2B), demonstrating that the fragrance is associated to the complexes. Additionally, the CBM::GQ20::SP-DS3-liposome/β-citronellol complex not subjected to the trigger was evaluated by GC-MS. We quantified around 50 μM of βcitronellol, suggesting that some fragrance might be absorbed in the liposomal membrane. Figure 6.2. Evaluation of protein and fragrance loss during ethanol injection method for production of the CBM::GQ20::SP-DS3-liposome complex by SDS-PAGE electrophoresis (A): (1) CBM::GQ20::SP-DS3; (2) CBM::GQ20::SP-DS3-liposomes; (3) nonanchored protein (CBM::GQ20::SP-DS3); (Mw) 5 μL GRS Unstained Protein Marker (GRISP); (B) GC-MS spectrum of nonencapsulated β-citronellol when separated from liposomes encapsulating fragrance and anchoring protein though a membrane with 100 kDa cut-off.
119 The stability of the CBM::GQ20::SP-DS3-liposome/β-citronellol complex and nonfunctionalized liposomes was evaluated over time in terms of size, polydispersity and surface charge, by dynamic light scattering (DLS). The liposomes were showed to be stable until 24 weeks (6 months) of storage at 4 °C. The nonfunctionalized liposomes were narrow and small size (around of 121 nm), with a monodisperse character (PDI ≈ 0.158) (Figure 6.3A), and a negative surface charge (≈ -36 mv) (Figure 6.3B). The CBM::GQ20::SP-DS3-liposome/β-citronellol complex also showed a narrow and small particle size (≈ 145 nm) and a negative surface charge (≈ -31 mV) (Figure 6.3D), revealing however higher polydispersity (PDI ≈ 0.285) (Figure 6.3C). The higher size and polydispersity index observed for CBM::GQ20::SP-DS3-liposome/β-citronellol complex might be associated with the presence of both encapsulated β-citronellol and anchored protein. Figure 6.3. Physicochemical characterization of nonfunctionalized liposomes (A, B) and functionalized liposomes encapsulating β-citronellol (C, D). Values represent the mean ± SD of 3 independent experiments.
120 3.2. Effect of acidic sweat solution on the properties of carbohydrate-binding module (CBM)-based complexes In this work, we evaluated the properties of CBM-fused proteins in the presence of an acidic sweat solution (trigger for the release of fragrances), evaluated in terms of size and polydispersity for the CBM::GQ20::SP-DS3-liposome/β-citronellol complex and in terms of binding affinity for the OBP::GQ20::CBM/ β-citronellol complex. The effect of acidic sweat solution on the physicochemical properties of the functionalized liposomes containing β-citronellol, such as the average diameter and the particles concentration, was evaluated using the Nanosight particle analyzer. As in nonfunctionalized liposomes, the addition of acidic sweat solution led to an increase of functionalized liposome size and to a reduction of the particle concentration (Figure 6.4A-C). In the presence of acidic sweat solution, the affinity of β-citronellol to OBP decreased, as highlighted by a decrease of the association constant (Ka = 3.06 ± 0.02 μM, Table 6.2). These results might indicate that acidic sweat solution is a good trigger for the release of β-citronellol from both approaches since it disturbs the physicochemical properties of the liposomes and affects the binding affinity properties of OBP, ensuring a controlled release without compromising the integrity of the systems (Table 6.2). Figure 6.4. Particles size (nm) and concentration (particles/mL) after 1 h incubation at 37 °C of nonfunctionalized and functionalized liposomes with an acidic sweat solution (pH 4.3 ± 0.2) (A); Graphical representation of concentration of particles versus size of particles in presence of buffer and
121 acid sweat solution (B); for a better visualization of graphic for functionalized liposomes, different scales were used (C).
122 Table 6.2. CBM-based complexes characterizationa Fusion protein Cargo container Cargo amount (β-citronellol) Cargo efficiency Effect of acidic sweat solution on container stability Association constant (Ka) Particles concentration Control Sweat effect Control Sweat effect OBP::GQ20::CBM OBP pocket 40 μM 100% 4.17 ± 0.05 μM 3.06 ± 0.02 μM - - CBM::GQ20::SP-DS3 liposomes core 1000 μM 95% - - 1.25E+09 particles/mL (Mode size 113 nm) 2.31E+08 particles/mL (Mode size 124 nm) aCargo container, cargo amount, cargo efficiency, association constant (Ka) of β-citronellol to OBP fusion-protein in buffer and acidic sweat solution, and effect of buffer and acidic sweat solution on the stability of liposomes functionalize with CBM::GQ20::SP-DS3 protein and encapsulating β-citronellol.
123 3.3. Functionalization of cotton fabrics with OBP::GQ20::CBM/β-citronellol and CBM::GQ20::SP-DS3liposome/β-citronellol complex 3.3.1. Efficiency of cotton functionalization The amount of CBM-based complexes containing β-citronellol at the surface of cotton fabrics was evaluated by dyeing the fabrics with a Coomassie blue solution (1%). The degree of fabrics functionalization is directly related with the color intensity measured in terms of K/S (Figure 6.5). The highest level of functionalization was observed when using 20 μM of proteins. The amount of CBMbased complexes at cotton surface, when using 20 μM, was determined by quantification of the unbound protein at 280 nm and by K/S evaluation (color staining levels) at maximum absorbance wavelength (at 610 nm). The absorbance data at 280 nm revealed a coating efficiency of 10.4 ± 0.1% and 12.9 ± 2.5%, for OBP::GQ20::CBM/β-citronellol and CBM::GQ20::SP-DS3-liposome/β-citronellol complex, respectively. The K/S analysis directly correlate the color intensity of the cotton samples with the amount of protein at the surface after functionalization The highest levels of functionalization obtained with 20 μM of protein were 28.1 ± 0.7% and 40.5 ± 0.4% for OBP::GQ20::CBM/β-citronellol and CBM::GQ20::SP-DS3-liposome/β-citronellol complex, respectively (Figure 6.5).
124 Figure 6.5. Amount of OBP::GQ20::CBM/β-citronellol and CBM::GQ20::SP-DS3-liposome/β-citronellol complex functionalized on cotton (%) determined by K/S evaluation. The values were obtained by subtracting the K/S value of buffer to the K/S value of each protein concentration. 3.3.2. Morphologic characterization of functionalized fabrics - SEM and EDS The functionalization efficiency by OBP::GQ20::CBM and by CBM::GQ20::SP-DS3-liposome complex was evaluated by SEM. The micrographs obtained for the CBM::GQ20::SP-DS3-liposome complex show the presence of macroscopic particles attached to the fabrics’ surface (Figure 6.6B), while the samples coated with OBP::GQ20::CBM reveal a more smooth and clean surface (Figure 6.6A). The quantification of the elements present in the samples was performed by Energy-Dispersive X-ray Spectroscopy (EDS) analysis and expressed in either weight or atomic concentration (Figure 6.6D). The results show the presence of nitrogen (N) on cotton coated with both OBP::GQ20::CBM and CBM::GQ20::SP-DS3liposomes complexes. The presence of liposomes on the cotton surface was confirmed by the presence of nitrogen as well as by the identification and quantification of phosphor (P), even in low amount, related to the phospholipid part of the CBM::GQ20::SP-DS3-liposomes complex (Figure 6.6D).
125 Figure 6.6. SEM photographs of cotton fabrics functionalized with OBP::GQ20::CBM (A) and CBM::GQ20::SP-DS3-liposome (B) and control (C). In D is presented the quantification of the elements present in cotton functionalized with OBP::GQ20::CBM and CBM::GQ20::SP-DS3-liposome complex and control by energy-dispersive X-ray spectroscopy (EDS) analysis. The data are expressed in either weight or atomic concentration. SEM images magnification ranging from 3,900x to 4,300x; scale bar of 20 µm. *Phosphor content in EDS analysis is above 1000 ppm (statistically not reliable).221 3.3.3. Quantification of fragrance release by Headspace-SPME/GC-MS trigger by acidic sweat solution For the design of the release systems, the implicit evaporation of the fragrance and the trigger mechanisms were considered.201 The aim of this work is the development of an efficient system for fragrance release from cotton. The experiments were performed at 37 °C to simulate the temperature increase of the skin after an external stimulus of stress or physical exercise, and an acidic sweat solution was applied to simulate the perspiration serving as trigger for fragrance release (Figure 6.1). The calibration curves were prepared using increasing concentrations of β-citronellol at the same conditions as the samples (Figure 6.7).