First insights into the binding mechanism and colour effect of the interaction of grape seed 11S globulin with malvidin 3-O-glucoside by fluorescence spectroscopy, differential colorimetry and molecular modelling

1 First insights into the binding mechanism and colour effect of the interaction of 1 grape seed 11S globulin with malvidin 3-glucoside by fluorescence spectroscopy, 2 differential colorimetry and molecular modelling 3 Francisco Chamizo-González1, Ignacio García Estevez2, Belén Gordillo1*, Elvira 4 Manjón2, M.T. Escribano-Bailón2, Francisco J. Heredia1, and M. Lourdes González- 5 Miret1. 6 1Food Colour & Quality Lab., Área de Nutrición y Bromatología. Facultad de Farmacia. 7 Universidad de Sevilla. 41012-Sevilla, Spain 8 2Grupo de Investigacin en Polifenoles, Facultad de Farmacia, Universidad de 9 Salamanca, Campus Miguel de Unamuno, E 37007 Salamanca, Spain 10 11 Francisco Chamizo-González: [email protected] 12 Ignacio García-Estévez: [email protected] 13 Belén Gordillo: [email protected] 14 Elvira Manjón: elvira[email protected] 15 M.T. Escribano-Bailón: [email protected] 16 Francisco J. Heredia: [email protected] 17 M. Lourdes Gonzalez-Miret: [email protected] 18 19 * Corresponding author: Belén Gordillo 20 Belén Gordillo 21 Food Colour & Quality Lab., Área de Nutrición y Bromatología. Facultad de Farmacia. 22 Universidad de Sevilla. 41012-Sevilla, Spain 23 Tel.: +34 9556760 24 e-mail: bg[email protected] 25 26 Manuscript Click here to view linked References 2 ABSTRACT 27 Recently, the search for alternative proteins endogenous to grapes to be used as wine 28 colour protecting agents became an important research trend. In this study, the molecular 29 interaction between the grape seed 11S globulin from winemaking by-product and 30 malvidin 3-glucoside was investigated by fluorescence, differential colorimetry and 31 molecular modelling. Fluorescence studies revealed the formation of grape seed protein- 32 pigment complex whose KS was 8.5x104 M-1 and binding sites, n=1.3. Malvidin 3- 33 glucoside showed darker and more vivid bluish colour of in the presence of 11S globulin, 34 suggesting the flavylium cation protection in a hydrophobic region of the protein. 35 Docking analysis and molecular dynamics simulation indicated that malvidin 3-glucoside 36 interacts mainly with the acidic subunit (40 kDa) of the 11S globulin monomer (60 kDa). 37 An average of two hydrogen bonds and Van der Wall forces were the main interaction 38 forces found for the protein-pigment complex, whose stability was confirmed by root- 39 means-square deviation. 40 41 Keywords: grape seed 11S globulin; winemaking by-product; malvidin 3-glucoside; 42 colour; fluorescence quenching; guided docking. 43 44 45 3 1. INTRODUCTION 46 Anthocyanins are natural flavonoid pigments responsible for orange-red to purple-blue 47 colours in many fruits, vegetables, and derived food products such as wines (Delgado- 48 Vargas, Jiménez,  Paredes-López, 2000). As potent antioxidants, anthocyanins are of 49 great interest for the food industry not only because they impact the sensory quality of 50 foods but also for their multiple health benefits associated with their consumption in the 51 diet (He  Giusti, 2010). 52 In red wines, anthocyanins are the 3-O-monoglucosides of five major anthocyanidins 53 (delphinidin 3-glucoside, cyanidin 3-glucoside, peonidin 3-glucoside, petunidin 3- 54 glucoside, and malvidin 3-glucoside) with malvidin 3-glucoside (mv3glc) and their 55 derivatives by far the most abundant pigments contributing for colour (Trouillas et al., 56 2016). The basic chromophore structure of anthocyanins consists in a flavylium cation 57 that shows a vivid red coloration in very acidic solutions (pH≤1). However, at moderately 58 acid pH values, as those found in wines (3.5-4.2), the flavylium cation is relatively 59 instable and very susceptible to decolouration and degradation against several factors 60 (Pina, Oliveira,  de Freitas, 2015). In wines, its colour stability is affected by pH, 61 anthocyanin chemical structure and concentration, exposure to temperature, oxygen, 62 light, enzymes, and other wine components or additives such as ascorbic acid, sugars, or 63 sulphites, among others (Escribano-Bailón, Rivas-Gonzalo,  García-Estévez, 2019). In 64 this regard, many winemaking techniques and oenological strategies have been developed 65 to preserve the anthocyanin stability and colour (Sacchi, Linda, Bisson, Douglas,  66 Adams, 2005; Soto Vázquez, Río Segade & Orriols Fernández, 2010). Moreover, natural 67 pigment-stabilizing mechanisms exist in wines that protect the coloured flavyliym cation 68 from the nucleophilic attack of water, pH changes, and decolouring agents; mainly 69 copigmentation interactions, but also certain associations with biopolymers including 70 4 polysaccharides or proteins (Boulton, 2001; Escribano-Bailón  Santos-Buelga, 2012; 71 Trouillas et al., 2016; Fernandes et al., 2021). 72 Over the last years, the researches on interactions between wine pigments and plant 73 proteins have been steadily increasing. This recent approach has provided valuable 74 information on the protection of anthocyanins through the interactions with different 75 protein types contributing to promote their application in the wine industry (Granato, 76 Ferranti, Iametti, & Bonomi, 2018; Marangon Vincenzi  Curione, 2019; Gordillo, 77 Chamizo-González, Gonzalez-Miret,  Herdia, 2021). 78 Plant seed-storage proteins include albumins, globulins, gliadins and glutenins fractions 79 of which globulins comprise one the major fraction. Currently, some studies have shown 80 that globulins from different vegetal sources present good functional properties to protect 81 the stability and antioxidant activity of anthocyanin pigments. Li et al. (2020) assessed 82 the complexation characteristics between different rice protein (RP) fractions and black 83 rice anthocyanins (ACN) by fluorescence spectroscopy at pH 3. This study concluded 84 that globulin-ACN complexes stablished strong binding interaction involving the 85 flavylium cation, with quenching constants larger than albumin-ACN complexes due to 86 the more hydrophobic areas of globulins. As well, the globulin-ACN complexes exhibited 87 greater DPPH (1,1-diphenyl-2-pycrylhydrazyl) and ABTS (2,2-azino-bis 3- 88 ethylbenzothiazoline-6-sulfonic acid) radical scavenging in comparison to RP albumin, 89 prolamin and glutelin due to the more flexible and unfolded structure of globulins 90 resulting in a better capability to react with the free radicals. On the other hand, Ren, 91 Xion,  Li (2019) compared the non-covalent binding mechanisms between cyanidin-3- 92 O-glucoside and two main soy protein fractions: β-conglycinin (7S) and glycinin (11S) 93 at pH 3, confirming the formation of spontaneous complexation processes between 94 globulins and the flavylium cation driven by electrostatic forces. In comparison, cyanidin 95 5 3-glucoside showed a stronger binding affinity toward 11S globulin owing to more 96 positive charges of 11S than 7S. Similarly, 7S and 11S globulins from soybean have also 97 demonstrated a protective effect on the stability of cyanidin 3-glucoside and to improve 98 its antioxidant capacity in simulated digestions. Again, the effects were stronger with the 99 11S globulin (Zheng, Zheng, Zhao, Yi,  Shengbao, 2021). 100 Of special interest for the wine industry are proteins endogenous to grape like those 101 obtained from grape seed wine by-products, which have been proposed as a sustainable 102 alternative to the use of other biopolymers subjected to more legal restrictions (i.e. animal 103 origin and synthetics) (Baca-Bocanegra, Nogales-Bueno, Hernández-Hierro, & Heredia, 104 2021; Gordillo et al., 2021; Mora-Garrido, Cejudo-Bastante, Heredia, & Escudero-Gilete, 105 2022). At this respect, the 3D structure of 7S and 11S globulins from Vitis vinifera L. 106 grape seed have been elucidated for the first time in our previous studies by combining 107 proteomic and computational techniques (Chamizo-González, Gordillo, & Heredia, 2021; 108 Chamizo-González, Heredia, Rodríguez-Pulido, González-Miret,  Gordillo, 2022). In 109 addition, we have confirmed by means of theoretical studies that both 7S and 11S grape 110 seed globulins could stablish different types of interactions (hydrogen bonding, alkyl and 111 π-π) with the main grape anthocyanin (mv3glc). However, such as interactions has been 112 scarcely studied and very little is still known concerning the real effect on the colour of 113 wine anthocyanins. Although a basic molecular interaction mechanism similar to 114 copigmentation has been suggested, many questions and experimental evidence remain 115 unclarified for the promising functionality of grape seed globulins. 116 Thus, this work aims to provide an improved understanding of the binding mechanism of 117 the interaction between the major grape seed storage protein (11S globulin, 11SGb) and 118 the major wine pigment (mv3glc) in relation to the effect on the anthocyanin colour. For 119 this purpose, fluorescence quenching in combination with colorimetric studies were 120 6 carried out in model solution to provide experimental evidence and fundamental 121 information about the molecular interaction (affinity constant and the stoichiometry of 122 the complex) as well as the potential colour effect. Complementary, molecular modelling 123 studies based on directed docking techniques were developed to assess the specific 124 binding site and the interaction forces of the protein-anthocyanin complex. This study is 125 expected to contribute to the potential use of plant proteins endogenous to grape as 126 potential colour protecting agents in wines. 127 2. MATERIAL AND METHODS 128 2.1. Reagents 129 Malvidin 3-glucoside (≥97 %) and Bovine Serum Albumin (BSA, ≥98 %) were purchased 130 from Extrasynthese (Genay, France). SDS-PAGE (sodium dodecyl sulphate- 131 polyacrylamide gel electrophoresis) reagents (Tris-HCl, dithiothreitol: SDS, 132 bromophenol blue, β-mercaptoethanol, glycerol, acrylamide, and bisacrylamide) were 133 purchased from Sigma-Aldrich (Steinheim, Germany). All other chemicals (ethanol, 134 citric acid, sodium citrate, sodium chloride, and trizma hydrochloride) were of analytical 135 grade and supplied by Panreac Química (Barcelona, Spain). 136 2.2. Extraction of grape seed globulins and purification 137 Seed globulins were extracted from defatted grape seed flour (industrial wine by-product) 138 supplied by ALVINESA Natural Ingredients, S.A. (Daimiel, Ciudad Real, Spain), 139 following the previously described method by Chamizo-González, et al. (2022) with 140 slight modifications. Globulin fraction (salt soluble) was extracted with 0.5 M Sodium 141 chloride (1:10 w/v) with constant stirring for 1 h at room temperature (25±0.2 C). The 142 obtained salt soluble fraction was centrifuged (8000 g for 10 min) and the flour residue 143 was submitted to the same process twice (each stirring for 30 min). The three supernatants 144 were combined, filtered through 0.45 μm nylon, and dialyzed during 48 h at 4 C against 145 7 ultrapure water using a Spectra/Por® 1 regenerated cellulose dialysis membrane 6-8 kDa 146 (Spectrum™ Labs.com Europe). 147 The globulin extract obtained (mainly comprised by 11SGb) was freeze-dried (lyophilizer 148 Cryodos-80, Telstar Varian DS 102) and stored at -20 °C for further analysis. 149 2.3. SDS-PAGE analysis of grape seed globulins and quantification of protein 150 subunits by image densitometry 151 The molecular weight and subunit composition of the grape seed globulin extract was 152 analysed under reducing conditions by SDS-PAGE using the Mini-Protean® 3 system 153 (BioRad, Hercules, CA, USA). Four replicates of the SDS-PAGE analysis were carried 154 out as follows: 10 mg of lyophilized protein extract was dissolved in 1 ml of distilled 155 water. Then, 150 µl of the aqueous protein extract was mixed with 50 µl of 0.02 M Tris- 156 HCl loading buffer pH 6.8 (containing glycerol 40% w/v, SDS 5% w/v, β- 157 mercaptoethanol 20%, and 0.01% Bromophenol Blue). Protein samples were then 158 denatured at 100 ˚C for 5 min. Samples (15 l) were loaded into 14% polyacrylamide gel 159 1.5 mm (acrylamide: bisacrylamide, 29:1) and electrophoresis was run at constant voltage 160 (170 V) for 50-60 min. Proteins were visualized after incubation with Coomassie staining 161 solution (40% v/v ethanol, 10% v/v acetic acid, 50% distilled water and 0.01 % 162 Coomassie blue R-250 w/v). Subsequently, the gels were decolorized in a solution of 40% 163 v/v ethanol and 10% v/v acetic acid while stirring until the bands were adequately 164 visualized. The standard molecular weight proteins (ranging from 20 to 200 kDa) used 165 were the PageRuler ™ unstained protein ladder (Thermo Fisher Scientific Inc., Vilnius, 166 Lithuania). 167 For quantification of 11SGb fraction and their respective subunits the protein bands were 168 analysed by image densitometry as described for other plant proteins (Mujoo, Trinh, & 169 Perry, 2003), using the software GelAnalyzer 2010a (www.GelAnalyzer.com). Gel 170 8 replicates (n=4) were scanned and the images were digitized and stored until needed for 171 quantitative analysis. The software GelAnalyzer 2010a allows display, store, and analyse 172 electrophoretic patterns covering all the main aspects of gel evaluation from automatic 173 lane detection to background subtraction methods, and calibration. A solution of 174 commercial standard BSA (1 mg/ml) was used as a protein standard for quantification 175 purposes. BSA solutions at increasing concentration (12.5, 25, 50, 100, and 200 µg/ml) 176 was loaded into each gel to obtain a calibration curve to quantify (µg) the different 177 globulin subunits. The proportions (%) of each globulin subunit relative to the sum of the 178 total subunits identified in the respective gel lane were also calculated. 179 2.4. Fluorescence quenching 180 The interaction between grape seed globulins and mv3glc was investigated using 181 fluorescence quenching measurements. The intrinsic fluorescence of proteins is due to 182 the presence of aromatic amino acids phenylalanine, tyrosine and tryptophan, the latter 183 being predominant in proteins (Lakowicz, 2013). Intrinsic fluorescence of samples was 184 carried out using a fluorescence spectrophotometer Perkin-Elmer LS 55 (Waltham, MA, 185 USA) in 1-cm quartz cuvettes at controlled temperature (25 °C ± 0.1). In all experiments, 186 proteins and mv3glc stocks solutions were prepared in 0.2 M citrate buffer (pH 3.5) with 187 the ionic strength adjusted with 0.5 M sodium chloride. The mixtures (final volume of 2 188 ml) were prepared at constant protein concentration (50 μM, mainly comprised of 11SGb) 189 and increasing concentrations of mv3glc (0-100 μM). The assay was performed in 190 triplicate. After mixing, the samples were transferred to the fluorimeter cell and the 191 emission spectra were measured. The excitation wavelength was 280 nm and the emission 192 spectrum was recorded between 290 and 450 nm. The excitation and emission slit widths 193 were both 5.5 nm, the scanning speed was 1200 nm/min, and the voltage was 700 v. After 194 each measurement, the cell was washed with ethanol and distilled water. 195 9 The fluorescence quenching constants were analysed by the Stern-Volmer equation (1) 196 (Wei et al., 2018). 197 𝐹0 𝐹= 1 + 𝐾𝑆𝑉 𝑥 [𝑄]= 1 + 𝐾𝑞𝑥 𝜏0 𝑥 [𝑄] (1) 198 where, F0 and F were the fluorescence intensities of proteins before and after the addition 199 of the quencher, [Q] is the molar concentration of mv3glc; KSV is the Stern-Volmer 200 quenching constant; Kq is the bimolecular quenching rate constant; and τ0 is the lifetime 201 of the fluorophore in the absence of quencher (10 -8 s) (Wei et al., 2018). 202 From the following equation (2) it is possible to calculate Kq (Rawel, Meidtner,  Kroll, 203 2005). 204 205 206 When Kq is greater than the diffusion-limited maximum extinction constant in water 207 (2x1010 M-1 s-1) indicates that static, and not dynamic, quenching was the main quenching 208 mechanism (Casanova et al., 2018). In this circumstance the Stern-Volmer Eq.(1) is 209 modified to the double logarithmic Stern-Volmer Eq. (3), where KS represents the “static 210 quenching constant” (Lakowicz, 2013): 211 212 213 Thus, for the static extinction mechanism, the number of binding sites n and the static 214 quenching constant KS can also be determined by linear regression from a plot of log (F0- 215 F/F) as a function of [Q]. The slope of the double logarithmic Stern-Volmer Eq. (3) 216 represents the number of binding sites n and the intersection represents the binding 217 constant. 218 𝐾𝑞=𝐾𝑠𝑣 𝜏0 (2) 𝑙𝑜𝑔 (𝐹0−𝐹) 𝐹= 𝑙𝑜𝑔𝐾𝑆+ 𝑛𝑙𝑜𝑔[𝑄] (3) 16 fluorescence data were analysed using the Stern-Volmer equations to determine the 368 quenching constants of the interaction. The fluorescence changes induced by mv3gcl 369 binding to the grape seed 11SGb are presented in Fig. 2B as the ratio F0/F versus the 370 mv3glc concentration at the maximum emission wavelength, from which KSV and Kq were 371 obtained from the slope of the regression curve. The value of the KSV was 7.7x103 M-1 372 with a 99 % confidence level and the value for Kq was 7.7x1011. As the Kq value was larger 373 than the maximal dynamic quenching constant in water (2×1010 M−1 s−1) it was confirmed 374 that mv3glc effectively quenched the intrinsic fluorescence of grape seed 11SGb by the 375 static mechanism predominantly caused by the formation of complexes. Similar 376 quenching mechanism has been reported for the interaction between cyanidin 3- 377 glcucoside and soybean 11S globulin (Chen et al., 2019; Ren et al., 2019; Dumitrascu, 378 Stănciuc, Grigore-Gurgu,  Aprodu, 2020). Thus, the static binding constant (KS) and 379 binding sites numbers (n) were calculated from Stern-Volmer Eq. (3) by plotting the log 380 (F0-F)/F versus log [mv3glc], which is showed in Fig. 2C. With a 99 % confidence level, 381 the value for the static quenching constant KS for the interaction between mv3glc and 382 grape seed 11SGb was 8.5x104 M-1 and the binding sites, n=1.3. These values were 383 consistent with the affinity constants values and binding sites described for complexes 384 between other proteins from vegetal (soy 11SGb) or animal origin (casein) with 385 anthocyanin compounds, most of them in the order of 104 M-1 (Chen et al., 2019; Li et 386 al., 2020). 387 3.3. Effect of grape seed 11SGb on mv3glc colour by differential tristimulus 388 colorimetry 389 Table 1 shows the influence of increasing concentrations of grape seed globulins (1.5, 390 2.5, and 5.0 mg/ml) on the CIELAB colour parameters (L*, a*, b*, b*, C*ab, hab) of 391 mv3glc solutions (10-4 M), assessed in 0.2 M citrate buffer (12 % ethanol at pH 3.5). As 392 17 observed, the increasing additions of the 11SGb extract resulted in a slight decrease of 393 lightness (L*) simultaneous to an increase of the chroma (C*ab) values, which mean that 394 mv3glc solutions became progressively darker and with more vivid colour at the protein 395 levels tested. These colorimetric effects reflect the shift of the anthocyanin equilibrium 396 toward more coloured forms in the presence of 11SGb suggesting a preferential binding 397 between such as grape seed proteins and the flavylium cation under simulated conditions 398 similar to those found in wines (12 % ethanol and pH 3.5). As well, a slight decrease of 399 the hue (hab) toward more negative values indicates that the blue component of colour 400 tended to increase in solutions, that is, mv3glc exhibited a more red-bluish tonality in the 401 presence of grape seed 11SGb. The slight positive changes observed in all the individual 402 colour parameters were in accordance with the gradual increases of the Total Colour (E) 403 of solutions (from 57 to 60 CIELAB u.), indicating that grape seed 11SGb produced a 404 global intensification of the mv3glc colour. Moreover, the ability of grape seed globulins 405 to modulate the colour characteristics of mv3glc seems to be significantly (p<0.05) 406 correlated with its concentration since the higher protein level tested, the significant 407 greater colour effects. However, the absolute differences in lightness, chroma and hue 408 (ΔL*, ΔC*ab and Δhab) calculated between the 11SGb-mv3glc solutions at the highest 409 protein level (5 mg/ml) respect to the mv3glc control solution (Table S1) denote that the 410 quantitative and qualitative colour changes produced were small (ΔL* =-2.4, ΔC*ab = 411 +1.7, and Δhab= -1 CIELAB u., respectively). 412 Thus, to establish whether the slight changes observed in the individual CIELAB 413 parameters were visually relevant, the colour differences (∆E*ab) between mv3glc 414 solutions and the same solutions containing increasing concentrations of grape seed 415 11SGb were calculated. Furthermore, by comparing the relative contributions of lightness 416 (%ΔL), chroma (%ΔC) and hue (%ΔH) that make up each colour difference (ΔE*ab) was 417 18 possible to assess which colour attribute was most influenced according to the protein 418 concentration. As observed in Fig. 3, the ΔE*ab values tended to be higher as the 419 concentration of grape seed globulins increased in the mv3glc solutions suggesting that 420 the colour effect becomes more noticeable depending on the protein content. Also, it was 421 confirmed that the colour enhancement was mostly due to quantitative changes (%L= 422 52-60 % and %∆C=33-35 % at 5 mg/ml of protein) and to a lesser extent to qualitative 423 changes (%∆H =7-13 % at 5 mg/ml of protein). However, the highest ΔE*ab values 424 observed at 2.5 and 5 mg/ml of protein ranged from 2.5 to 3.5 CIELAB u., which are 425 considered values close to the visual threshold to clearly perceive colour differences by 426 average non-trained observers, according to Martínez, Melgosa, Pérez, Hita, & 427 Negueruela (2001). Thus, the magnitude of the colour changes observed both in the 428 individual parameters and on the colour differences was slightly visually relevant, and 429 comparatively less notable than the colour impact reported for other compounds (as 430 certain phenolic copigments) when tested in similar conditions (Gordillo et al., 2012; 431 Gordillo et al., 2015; Rivero et al., 2022). These findings encourage developing further 432 and new investigations to better optimize the potential technological use of grape seed 433 globulins as colour protecting agents in wines or in the food industry. 434 3.4. Guided-docking study and analysis between grape seed 11SGb and mv3glc 435 Fluorescence quenching and colorimetric studies provided evidence of the formation of 436 a grape seed protein-wine anthocyanin complex resulting in the protection of the 437 flavylium cation against hydration reaction a pH 3.5 and in positive effects on mv3glc 438 colour. With the aim of supporting from a molecular point of view the experimental 439 interactions observed, computational studies were developed. In particular, a guided- 440 docking study was carried out between one of the grape seed 11SGb monomer identified 441 by mass spectrometry (F6HZK3, 60 kDa) in our previous study (Chamizo et al., 2022) 442 19 and mv3glc in order to improve the accuracy in the binding affinity prediction of the 443 interaction between grape seed proteins and wine anthocyanins. 444 For this purpose, this new docking study was carried out selecting specifically the major 445 hydrophobic region of the protein monomer (60 kDa) determined in our previous 446 computational results (Chamizo et al., 2022), as the most susceptible protein site to 447 interact with mv3glc. 448 Nine possible ligand conformations in the major hydrophobic protein environment were 449 obtained after each run in the AutoDock Vina software and the lowest energy 450 conformation (-6.8 Kcal/mol) was chosen as the most favourable conformation in the 451 binding site. This conformation is illustrated in Fig. 4A which shows the most favourable 452 specific binding site of the 11SGb monomer with the mv3gcl into the major hydrophobic 453 region of the protein (indicated in orange). As well, the binding model was represented 454 in two dimensions in Fig. 4B where the interactions established between the protein and 455 mv3glc are shown. As observed, the amino acids involved in the interaction were mainly 456 Gly 151 and Gln 133, which establish hydrogen bond with the hydroxyl groups of the 457 glucose and the A ring of mv3glc, respectively. Moreover, the aromatic amino acid 458 Phe153 establishes π-π and alkyl hydrophobic interactions with both B and C rings of 459 mv3glc (binding distance about 4.1 and 4.4 Å). Other interactions such as Van der Waals 460 forces also are present in the protein-ligand interaction. Such as hydrophobic interactions 461 are similar to those widely described for different copigmentation associations between 462 wine copigments and anthocyanins, according to Trouillas et al., (2016). Our results also 463 agreed with those of protein-anthocyanin interactions involving 11SGb from other 464 vegetal sources (soy proteins), which have confirmed that aromatic amino acids such as 465 tryptophan are responsible of the non-covalent binding with the pigment (Dumitriascu et 466 al., 2020). 467 20 3.5. Stability analysis of the grape seed 11SGb-mv3glc complex by molecular 468 dynamics simulation 469 The stability of the binding conformation and mode for the 11SGb-mv3glc complex was 470 assessed by MD simulation during 10 ns in a solvated and equilibrated system. In 471 addition, the stability of the 11SGb and the mv3glc were individually assessed for 472 comparative purposes. One of the most commonly used parameters in MD to analyse the 473 conformational stability of a protein-ligand complex is the RMSD parameter, in this case 474 applied to C-α atoms of the protein backbone. In general, low values of RMSD with 475 constant fluctuations during MD simulation time indicate that the system is stable while 476 high RMSD values are typical of unstable systems (Wu, et al., 2021; Zhao, et al., 2022). 477 The plot of RMSD values during the MD simulation time for the three systems (11SGb, 478 mv3glc, and the 11SGb-mv3glc complex) is shown in Fig. 5A. The RMSD values 479 obtained for the 11SGb-mv3glc complex ranged from 0.20 to 0.50 nm showing small 480 fluctuations. In the case of the 11SGb alone (red line), the values of RMSD ranged from 481 0.2 to 0.65 nm and for the mv3glc (blue line) between 0.10 and 0.20 nm. Thus, the 11SGb- 482 mv3glc complex exhibited lower RMSD values than the protein alone (11SGb) 483 suggesting that the binding of the mv3glc to the grape seed protein improved the 484 conformational stability of the system. Furthermore, the lowest RMSD values found for 485 the mv3glc alone indicate that the pigment is stable in the protein-binding site during the 486 simulation. These conformational changes can be better visualized in Fig. 1S, and the Fig. 487 5B, where the protein-pigment interaction is showed with more detail during the last 488 period of the MD simulation (7, 8, 9, and 10 ns). 489 The number of hydrogen bonds formed by the 11SGb with mv3glc was calculated and 490 plotted versus MD simulation time (Fig. 5C). Results showed that the protein established 491 between 0 and three hydrogen bonds with the pigment during the MD simulation, with 492 21 an average value of two hydrogen bonds. The analysis of MD simulation results using the 493 Discovery studio software showed that the main amino acids involved in the interaction 494 with hydroxyl groups of the A ring of mv3glc trough hydrogen bonds were Gln 133 and 495 Glu 82, which agreed with the docking results for the former. These findings suggest that 496 Gln 133 residue of the grape seed 11SGb could have a key role in the stabilisation of 497 mv3gcl. Such as amino acid residues have also been reported to participate the interaction 498 of heated soy proteins with anthocyanins from cornelian cherry fruits (Dumitrascu et al., 499 2020). 500 CONCLUSIONS 501 Fluorescence quenching in combination with colorimetric studies confirmed that the 502 grape seed 11SGb interacts with mv3gcl through static binding mechanism contributing 503 to positively modulate the colour characteristics of the major grape and wine anthocyanin. 504 The colorimetric benefits achieved (mainly quantitative and to a lesser extent qualitative) 505 were dependent on the protein concentration and slightly visually discernible. The 506 formation of a stable 11SGb-mv3glc complex in the most hydrophobic site of the protein 507 driven by hydrogen bounds and Van der Walls forces was suggested by the molecular 508 modelling studies. Specifically, the acidic subunit of 11SGb monomer mainly involved 509 in the interaction was found as a one of the major polypeptide of the grape seed globulin 510 fraction, which could be of particular interest for colour protection applications in the 511 food industry and especially in winemaking sector. 512 513 Acknowledgments 514 Authors thank the assistance of the technical staff of Biology Service (SGI, Universidad 515 de Sevilla, Spain) and the Scientific Computation Center of Andalusia (CICA) for the 516 computing services they provided. 517 22 Funding sources 518 This research was financially supported by the Ministerio de Economía y Competitividad, 519 Spain, Gobierno de España (Project PID2021-127126OB-C2 and FPI grant PRE2018- 520 087184). E. 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Figure 1 Click here to access/download;Figure(s);Chamizo_11SGbMv_Figure 1.docx Figure 2. A) B) Figure 2 Click here to access/download;Figure(s);Chamizo_11SGbMv_Figure C) Figure 3. Rep 1 Rep 2 Rep3 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 E*ab %  H %  C  %  L 54.1% 38.6% 59.9% 56.3% 52.2% 38.8% 38.3% 47.1% 42.1% 49.9% 32.9% 33.8% 34.8% 12.4% 10.6% 11.5% 7.2% 9.9% 13.0% 49.3% 50.6% 35.5% 48.5% 61.2% 11SGb_ 1.5 mg/mL 11SGb_ 2.5 mg/mL 11SGb_ 5.0 mg/mL Figure 3 Click here to access/download;Figure(s);Chamizo_11SGbMv_Figure 3.docx Figure 4 A) B) Figure 4 Click here to access/download;Figure(s);Chamizo_11SGbMv_Figure Figure 5. A) B) Figure 5 Click here to access/download;Figure(s);Chamizo_11SGbMv_Figure 5.docx C) Table S1. Absolute colour, lightness, chroma, and hue differences (ΔE*ab, ΔL*, ΔC*ab, Δhab) calculated between the 11SGb-mv3glc solutions with respect to the mv3gcl control solution (10-4 M, n=3), at increasing protein concentration (1.5, 2.5, and 5.0 mg/ml). [Mv3glc + 11SGb]- Mv 3 glc 1.5 mg/ml 2.5 mg/ml 5.0 mg/ml E*ab 1.78±0.06 a 2.56±0.01b 2.85±0.29b L* - 1.31±0.13a - 1.59±0.01a - 2.14±0.29b C*ab + 0.34±0.01a + 1.81±0.02b + 1.66±0.15b hab + 1.34±0.06a + 1.00±0.06b + 1.02±0.04b Different letters in the same row indicate significant differences (p<0.05, Tukey's test) Table S1 Fig. 1S. Dynamic molecular simulation Figure S1 Click here to access/download;Supplementary Material;Chamizo_11SGbMv_Supp Material_FigS1.pptx Figure S2. Absorption spectra (A.U.) of the mv3glc model solutions (10-4 M) in the presence of increasing concentrations of the11SGb extract (0, 1.5, 2.5, and 5.0 mg/ml). The spectra of mv3glc solutions were corrected in the visible range by the absorbance spectra of 11SGb solutions at increasing concentrations. 0.0 0.2 0.4 0.6 0.8 1.0 360 401 442 483 524 565 606 647 688 729 770 A.U. (nm) 11SGb 0 mg/ml 11SGb 1.5 mg/ml 11SGb 2.5 mg/ml 11SGb 5 mg/ml Figure S2 Click here to access/download;Supplementary Material;Chamizo_11SGbMv_Supp Material_FigS2.docx