Rúa Jena o de la Fuen e, s/n –Campus Vida – Uni e sidade de San iago de Compos ela -15782 San iago de Compos ela – ciqus.usc.es
Sho oligoalanine helical pep ides o sup amolecula
nanopo e assembly and p o ein cy osolic deli e y
Ma a Pazo, Giulia Salluce, I ene Los alé-Seijo, Ma isa Juanes, F ancisco Gonzalez,
Rebeca Ga cia-Fandiño and Ja ie Mon eneg o
Suppo ing in o ma ion
Copy igh in o ma ion:
© 2021 The Au ho (s). Published by he Royal Socie y o Chemis y
S1
Sho Oligoalanine Helical Pep ides o Sup amolecula
Nanopo e Assembly and P o ein Cy osolic Deli e y
Ma a Pazo, Giulia Salluce, I ene Los alé-Seijo, Ma isa Juanes, F ancisco Gonzalez,
Rebeca Ga cia-Fandiño and Ja ie Mon eneg o*
e-mail o: [email p o ec ed]
Suppo ing In o ma ion
Table o con en s
1. Suppo ing Figu es .................................................................................................................... 2
2. Expe imen al P ocedu es ........................................................................................................ 17
2.1. Ma e ials .............................................................................................................................. 17
2.2. Abb e ia ions ....................................................................................................................... 18
2.3. Gene al p o ocol o he SPPS ........................................................................................... 18
2.3.1. Syn hesis o pep ide MP1 ............................................................................................ 19
2.3.2. Syn hesis o pep ide CF-MP1 ...................................................................................... 19
2.3.3. Syn hesis o pep ide TM-MP1 ..................................................................................... 19
2.3.4. Syn hesis o pep ide MP2 ............................................................................................ 19
2.3.5. Syn hesis o pep ide P3 ................................................................................................ 19
2.3.6. Syn hesis o pep ide A g8 ............................................................................................. 19
2.3.7. Syn hesis o pep ide GALA ......................................................................................... 20
2.3.8. Syn hesis o pep ide L17E ........................................................................................... 20
2.3.9. Syn hesis o Pep-1 ........................................................................................................ 20
2.3.10. Syn hesis o d TAT ..................................................................................................... 20
2.4. Liposome p epa a ion ......................................................................................................... 20
2.5. ANTS/DPX assay ................................................................................................................. 21
2.6. Dex an elease in esicles .................................................................................................. 21
2.7. Ci cula Dich oism ............................................................................................................. 22
2.8. DLS expe imen s ................................................................................................................. 22
2.9. FRET and luo esence expe imen s ................................................................................... 22
2.10. Compu a ional me hods ................................................................................................. 23
2.11. Plana lipid bilaye eco dings ...................................................................................... 23
2.12. Cell cul u e expe imen s ................................................................................................. 24
2.12.1. Cell lines and cul u e ................................................................................................ 24
2.12.2. Cellula up ake and endosomal elease o dex an ................................................... 24
2.12.3. Cell iabili y assay (MTT) ....................................................................................... 25
2.12.4. Sapo in expe imen s ................................................................................................. 25
2.12.5. C e ecombinase exp ession and pu i ica ion .......................................................... 25
2.12.6. C e ecombinase deli e y expe imen s .................................................................... 25
2.12.7. An ibody deli e y in cell cul u e .............................................................................. 26
2.12.8. CF-labelling and deli e y o IgG ............................................................................. 26
2.12.9. Pu i ica ion and deli e y o GST-NLS-GFP ............................................................ 26
2.13. Ex i o co nea expe imen s ............................................................................................ 27
2.13.1. Mu ine co neas cul u e ............................................................................................. 27
2.13.2. An ibody deli e y in co nea ..................................................................................... 27
2.13.3. Li e/Dead S aining ................................................................................................... 27
2.14. Sou ces o Figu e 6 able .............................................................................................. 27
3. Suppo ing Re e ences ............................................................................................................ 28
S2
1. Suppo ing Figu es
Figu e S1. Ci cula dich oism spec a o MP1, MP2 and P3 in HKR bu e (g ay), Liposomes ( ed)
(L/P a io: 12) and TFE (o ange). Measu emen s we e done a 40 °C. See also Fig. 1d.
Figu e S2. CF-MP1 agg ega es in solu ion. A) Emission spec a o CF-MP1 (λex = 494 nm) in aqueous
solu ion a di e en concen a ions. B) Maximum luo escence in ensi y a di e en concen a ions C)
Wa eleng h o he peak o maximum luo escence a di e en concen a ions.
S3
Figu e S3. Vesicle elease expe imen s. A) Kine ic luo escen aces o LUVsÌANTS/DPX a e addi ion o MP1 (a = 25 s). T i on X-100 was added a 225 s o
comple e esicle lysis and no maliza ion. B) Dose esponse cu es o dex an elease in neu al POPC esicles (LUVsÌDex an) a di e en MP1 concen a ions. The 10
kDa FITC-Dex (g een) and 40 kDa TM-Dex (pink) a e shown wi h emp y squa es (pH = 7.5) and illed ci cles (pH = 5.5) espec i ely. C) DLS measu emen s o esicles in
he p esence o MP1. Rep esen a ion o pho on coun a e (an indica o o measu emen quali y) and size (hyd odynamic adius, indica i e o esicle size and s abili y). E o
ba s indica e he s anda d de ia ion o h ee eplica es calcula ed by equa ion S5 o DLS expe imen s. In all cases he bu e was MES 10 mM wi h NaCl 150 mM.
Expe imen s we e done wi h neu al POPC o anionic POPC/POPG esicles a ei he pH 7.5 o pH 5.5 as indica ed a he op o he cha s.
S4
Figu e S4. Molecula dynamics simula ion. Top: Snapsho a = 70 ns o he CG-MD simula ion o 31
uni s o pep ide MP1 in e ac ing wi h a memb ane composed by DPPC:DPPE (9:1) and
DPPC:DPPE:DPPS (3:5:2), in absence o elec ic ield. Middle: De ail o he inse ion o he pep ides in
he memb ane by ancho ing and o ien ing he Leu (o ange) en iched hyd ophobic N- e minus o pep ide
MP1 owa ds he hyd ophobic co e o he lipid bilaye , and he a ginine (blue) ca ionic esidues exposed
o he sol en . Down: De ail o he op iew o he ensemble o MP1 in e ac ing wi h he memb ane,
unde an ex e nal elec ic ield o 0.03 V/nm applied ac oss he memb ane ( = 300 ns, om Figu e 5). An
ini ial po a ion o he memb ane can be in e ed.
S5
A)
B)
Figu e S5. Time lapse o dex an cy osolic elease in HeLa cells. A) HeLa cells we e incuba ed wi h
0.25 mg/mL o Alexa488-Dex an (10 kDa, g een) in he p esence o 5 µM o TM-MP1 ( ed) o 1 h in
DMEM wi hou FBS. Cells we e washed and incuba ed in DMEM wi h FBS o 1 hou , 2 hou s o 3
hou s be o e imaging. Top ows: 20x magni ica ion; bo om ows: 60x magni ica ion. Each se o images
shows: me ge o b igh ield and TM-MP1 ( ed; uppe le ), dex an luo escence (g een; uppe igh ),
me ge o b igh ield and dex an (lowe igh ), me ge o TM-MP1, dex an, and b igh ield (lowe le ).
Scale ba s = 50 µm. B) HeLa cells we e incuba ed wi h 0.25 mg/mL o FITC-Dex an (10 kDa, g een), in
he p esence o 5 µM TM-MP1 ( ed) o 1 h in DMEM wi hou FBS. Cells we e washed and incuba ed in
DMEM wi h FBS o 3 h and hen s ained wi h LysoT acke Deep Red (100 nM, 30 min, shown in blue),
washed and obse ed a he mic oscope. Scale ba s = 25 µm.
S6
Figu e S6. Dex an elease in he p esence o se um o endocy osis inhibi o s. A) HeLa cells we e
incuba ed o 1 h wi h a solu ion o 0.25 mg/mL 10 kDa Alexa488-Dex an (g een) and he indica ed
concen a ions o MP1 in DMEM s ock ( op ow) o in DMEM supplemen ed wi h 10% FBS (bo om
ow). Cells we e hen washed and incuba ed o 3 hou s wi h DMEM supplemen ed wi h 10% FBS be o e
con ocal imaging. Cells wi h di use signal we e coun ed and exp essed as pe cen age. G aph shows
mean o h ee expe imen s ± SD. B) HeLa cells we e incuba ed wi h 0.25 mg/mL 10 kDa TM-labelled
dex an and 30 µM pep ide MP1 in he p esence o he indica ed inhibi o s o 1 h. A e incuba ion, he
numbe o cells wi h cy osolic dex an was coun ed and no malized o he con ol wi h 30 µM pep ide in
he absence o inhibi o s (100%). Da a is p esen ed as mean o h ee independen expe imen s ± SD.
Rep esen a i e mic og aphs o each condi ion a e shown a he bo om o he image ( op ow: dex an
luo escence; bo om ow: me ge o dex an luo escence ( ed) wi h b igh ield). Scale ba s = 100 µm.
S7
Figu e S7. Func ional P o ein Cy osolic Deli e y. A) HeLa cells we e incuba ed wi h 0 (g ey ba s) o
10 µg/mL ( ed ba s) sapo in in he p esence o inc easing concen a ions o pep ides A g8 o GALA o 1
h; medium was hen eplaced and cells u he incuba ed o 6 h be o e measu ing iabili y by MTT
assay. B, C) Analogous expe imen in HeLa (B) and A549 (C) cells by incuba ion wi h MP1 (1 h) and
u he incuba ion o 24 h be o e iabili y quan i ica ion by MTT assay.
Figu e S8. C e ecombinase deli e y e iciency by di e en pep ides. A) and B) C e- epo e HeLa
cells we e ea ed wi h 1 µM C e ecombinase and 2.5, 5, 10, 20 µM o he indica ed pep ides o 4 h, and
he numbe o cells ha swi ched om ed o g een (GFP) luo escence due o he ecombinase ac i i y
quan i ied by low cy ome y 3 days a e incuba ion. The able indica es he pe cen o GFP-exp essing
cells ± SD (n = 3) o each pep ide and concen a ion. C) TM-MP1 and C e o m agg ega es. HeLa-C e
epo e cells we e incuba ed wi h 1 µM C e ecombinase and 20 µM TAMRA-labelled MP1 (TM-MP1)
o 4 h, and cells imaged 3 days la e . Bo h TAMRA (pep ide) and dsRED (exp essed by cells) a e shown
in ed, GFP in g een and Hoechs -s ained nuclei in blue. A ows indica e luo escen pep ide agg ega es
as de ec ed by he TAMRA luo escence, which may be esponsible o he lowe up ake a high
concen a ions.
S8
Figu e S9. Deli e y o GST-NLS-GFP and IgG-CF in o HeLa cells. A) HeLa cells we e incuba ed in
DMEM wi h GST-NLS-GFP (10 µM; shown in g een) in he absence o p esence o 30 µM MP1 o 1 h,
washed and u he incuba ed o 90 min in DMEM supplemen ed wi h 10% FBS be o e con ocal
imaging. Nuclei we e s ained wi h Hoechs (blue). A ound 15% o he cells showed nuclea dis ibu ion
o GST-NLS-GFP. B) HeLa cells we e incuba ed wi h IgG-CF (1 mg/mL, g een) in he p esence o
absence o 30 µM MP1 o 1 h, in he same condi ions as in A. Scale ba s = 50 µm.
IC50
HeLa
VERO
A549
ARPE-19
MP1
126.6 µM
116.2 µM
137.5 µM
133.4 µM
Figu e S10 MTT iabili y assays. Viabili y was de e mined in HeLa (A), Ve o (B), A549 (C) and
ARPE-19 (D) cells. Cells we e incuba ed wi h di e en concen a ions o MP1 (0, 1, 2, 4, 8, 16, 32, 62.5,
125, 250, 500, 1000 µM) in DMEM o 1 h, hen he solu ions we e emo ed and eplaced wi h comple e
medium. A e 24 hou s, cells we e incuba ed o 3 hou s wi h medium supplemen ed wi h 0.5 mg/mL
MTT e azolium sal , and he medium was emo ed be o e solubilizing he o mazan sal wi h DMSO.
Cells iabili y was hen quan i ied by abso bance. Da a was no malized o he un ea ed con ol (100 %).
Da a is shown as mean o i e eplica es ± SD.
S15
Figu e S23. d TAT cha ac e iza ion. RP-HPLC [Agilen SB-C18 column, H2O (0.1% TFA)/ CH3CN
(0.1% TFA) 95:5→5:95 (0→20 min)] (R 11.5 min) abso bance a 222 nm and ESI-MS o d TAT.
S16
Video S1. De ail o a CG-MD simula ions ajec o y (5 ns in o al) highligh ing he ini ial in e ac ion o
MP1 wi h a lipid bilaye composed by DPPC:DPPE (9:1) and DPPC:DPPE:DPPS (3:5:2). As i can be
no iced, al hough he p elimina y in e ac ion o MP1 wi h he memb ane is led by elec os a ic
in e ac ions be ween he A g esidues and he pola heads o he lipids, he pep ide quickly o ien s he Leu
en iched hyd ophobic N- e minus o MP1 owa ds he hyd ophobic co e o he lipid bilaye , and he
a ginine ca ionic esidues emain exposed o he sol en . CG esidues a e ep esen ed in yellow (Ala),
o ange (Leu) and blue (A g).
S17
2. Expe imen al P ocedu es
2.1. Ma e ials
Comme cially a ailable Rink Amide- esin ChemMa ix, Fmoc-L-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L-
A g(pb )-OH, iisop opylsilane (TIS), Diisop opyle hyl amine (DIEA), diisop opylca bodiimide (DIC),
and 5-(N-E hyl-N-isop opyl)amilo ide (EIPA) we e ob ained om Sigma-Ald ich. T i luo oe hanol,
1,1,1,3,3,3-Hexa luo oisop opanol (HFIP) and chlo p omazine we e pu chased om TCI. Egg yolk L-α-
phospha idylcholine, 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine in chlo o o m (POPC), 1-
palmi oyl-2-oleoyl-sn-glyce o-3-phosphoglyce ol sodium sal in chlo o o m (POPG), 16:0-12:0 1-
palmi oyl-2-{12-[(7-ni o-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glyce o-3-
phosphoe hanolamine (NBD-PE), 1,2-dipalmi oyl-sn-glyce o-3-phosphoe hanolamine-N-(7-ni o-2-1,3-
benzoxadiazol-4-yl) (PE-NBD) and 1,2-diphy anoyl-sn-glyce o-3-phosphocholine (DPhPC) we e
pu chased om A an i Pola Lipids. E hyl(hyd oxyimino)cyanoace a e (Oxyma), me hyl-β-cyclodex in,
and 5-ca boxy e ame hyl hodamine (TAMRA) we e a ailable om Ca bosyn h. N-HATU was p o ided
by Glen ham li e sciences. N-HBTU was ob ained om I is. Pep ide syn hesis g ade N,N-
dime hyl o mamide was pu chased om Scha lau. Dynaso e was om EMD Millipo e Co po a ion. All
he o he sol en s we e HPLC g ade, pu chased om Sigma-Ald ich® o Fishe Scien i ic®, and used
wi hou u he pu i ica ion.
Dulbecco’s Modi ied Eagle’s Medium (4500 mg/L glucose, L-glu amine, sodium py u a e and
sodium bica bona e) was ob ained om Gibco. Pu omycin was pu chased om Enzo Li e Sciences.
Ames’ medium, Alexa Fluo 488 – Dex an (10 kDa), Te ame hyl hodamine – Dex an (10 kDa),
luo escein – Dex an (70 kDa), and LysoT acke Deep Red we e acqui ed om The mo Fishe . FITC –
Dex an (40 kDa), Sapo in, Dulbecco's Modi ied Eagle's Medium/Nu ien F-12 Ham (4500 mg/L
glucose, L-glu amine, sodium py u a e and sodium bica bona e), human IgG (#I4506) and an i-nuclea
po e complex p o eins an ibody Mab414 (Mouse monoclonal, clone 414, #N8786) we e pu chased om
Sigma-Ald ich.
A mic owa e assis ed pep ide syn hesise (Libe y Li e, CEM) was used o p epa e he pep ide
acco ding o s anda d me hods de eloped by he manu ac u e s in ol ing diisop opylca bodiimide (DIC)
0.5 M in DMF as ac i a o and e hyl(hyd oxyimino)cyanoace a e (Oxyma) 1 M in DMF as ac i a o base.
High-pe o mance liquid ch oma og aphy coupled wi h mass spec ome y (HPLC-MS) analyses we e
ca ied ou on Agilen Technologies 1260 In ini y II associa ed wi h a 6120 Quad upole LC-MS using an
Agilen SB-C18 column o on DIONEX Ul ima e 3000 U-HPLC+ (The mo Scien i ic) wi h an Acclaim
RSLC 120-C18 column wi h Sol en A:Sol en B g adien s be ween 5:95 (Sol en A: H2O wi h 0.1%
TFA; Sol en B: CH3CN wi h 0.1% TFA). High-pe o mance liquid ch oma og aphy (HPLC) semi-
p epa a i e pu i ica ion was ca ied ou on Jasco LC-4000 wi h an Agilen Eclipse XDB-C18 column.
Accu a e mass de e mina ions (HR-MS) using ESI-MS we e pe o med on a B uke Mic oTo mass
spec ome e .
Ci cula Dich oism (CD) measu emen s we e pe o med wi h a Jasco J-1100 CD Spec ome e
equipped wi h a Jasco MCB-100 Mini Ci cula ion Ba h o empe a u e con ol.
Vesicles we e p epa ed wi h a Mini-Ex ude om A an i Pola Lipids (po e size 100 nm). Sample
incuba ion was pe o med in 500 µL eppendo ials. Fluo escence measu emen s we e pe o med wi h a
Fluo oMax-2 spec o luo ome e (Jobin-Y on Spex) equipped wi h a s i e and a empe a u e con olle .
DLS measu emen s we e pe o med in a Mal e n Ze asize Nano ZSP using s anda d disposable
cu e es.
Fo he acquisi ion o cell mic oscopy images a Nikon Eclipse Ti-E epi luo escence mic oscope wi h
an Ando Zyla 4.2 digi al came a, o a D agon ly con ocal spinning-disk on a Nikon Eclipse Ti-E
equipped wi h a Ando Zyla 4.2 PLUS sCMOS digi al came a we e used. Images we e p ocessed wi h
FIJI.S1 3D images we e econs uc ed om he di e en indi idual con ocal planes wi h Ima is 9.0.0
so wa e (Ox o d ins umen s).
A Tecan In ini e F200P o mic opla e eade was used o di ec ly measu e in Cos a cell cul u e 96-
well pla es UV-Vis abso bance o he MTT iabili y assays. Flow cy ome y was pe o med on a Gua a
easyCy eTM cy ome e and da a analysed wi h InCy e so wa e included in Gua aSo 3.2 (Millipo e).
S18
2.2. Abb e ia ions
Aa: Amino acid; A g: A ginine; BSA: bo ine se um albumin; Calcd: Calcula ed; CF:
Ca boxy luo escein; CF-MP1: oligoalanine MP1 labelled wi h ca boxy luo escein a N- e minus; CPZ:
Chlo p omazine; DCM: Dichlo ome hane; Dex: dex an; DIC: diisop opylca bodiimide; DIEA: N,N-
Diisop opyle hylamine; DMF: N,N-Dime hyl o mamide; DMEM: Dulbecco’s Modi ied Eagle’s Medium;
DPPC: dipalmi oyl phospha idylcholine; DPhPC: 1,2-diphy anoyl-sn-glyce o-3-phosphocholine; DPPE:
dipalmi oyl phospha idyle hanolamine; DPPS: dipalmi oyl phospha idylse ine; Dyn: Dynaso e; EGFP:
enhanced g een luo escen p o ein; EIPA: 5-(N-E hyl-N-isop opyl)amilo ide; FDA: luo escein diace a e;
GST: glu a hione-S- ans e ase; HFIP: 1,1,1,3,3,3-Hexa luo o-2-p opanol; HRMS (ESI): High esolu ion
mass spec ome y (elec osp ay ioniza ion); IPTG: Isop opyl β-D-1- hiogalac opy anoside; LB:
Lysogeny b o h; Leu: Leucine; Lys: Lysine; LUV: la ge unilamella esicles; MbCD: me hyl-β-
cyclodex in; MES: 4-Mo pholinee hanesul onic acid; Leu: Leucine; Lys: Lysine; MP1: minimal pep ide
1; MP2: pep ide 2 wi h aligned leucines; MOPS: 3-(N-Mo pholino)p opanesul onic acid; M : 4-
Me hyl y il; MTT: 3-(4,5-dime hyl-2- hiazolyl)-2,5-diphenyl e azolium b omide; NBD-PE: 16:0-12:0
1-palmi oyl-2-{12-[(7-ni o-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glyce o-3-
phosphoe hanolamine; N-HATU: N-[(Dime hylamino)-1H-1,2,3- iazolo[4,5-b]py idine-1ylme hylene]-
N-me hylme hanaminium-hexa luo ophospha e N-oxide; N-HBTU: N-[(1HBenzo iazol-1-yl)-
(dime hylamino)me hylene]-N-me hylme hanaminium hexa luo ophospha e N-oxide; NLS: Nuclea
Localiza ion Signal; P3: longe oligoalanine pep ide; PE-NBD: 1,2-dipalmi oyl-sn-glyce o-3-
phosphoe hanolamine-N-(7-ni o-2-1,3-benzoxadiazol-4-yl); Pb : 2,2,4,6,7-
Pen ame hyldihyd obenzo u an-5-sul onyl; PBS: Phospha e-Bu e ed Saline; POPC: 1-palmi oyl-2-
oleoyl-sn-glyce o-3-phosphocholine; PI: p opidium iodide; POPG: 1-palmi oyl-2-oleoyl-sn-glyce o-3-
phosphoglyce ol; SPPS: solid phase pep ide syn hesis; SV40: Simian Vi us 40; TAMRA: 5-
ca boxy e ame hyl hodamine; TFE: T i luo oe hanol; TIS: T iisop opylsilane; TM-MP1: oligoalanine 1
labelled wi h TAMRA a N- e minus; TNBS: 2,4,6-T ini obenzenesul onic acid;.
2.3. Gene al p o ocol o he SPPS
All pep ides we e syn hesized by au oma ed o manual Fmoc solid-phase pep ide syn hesisS2 using
Rink Amide ChemMa ix esin (loading 0.5 mmol/g). Fo manual syn hesis, he esin (0.5 mmol) was
swelled in DMF (pep ide syn hesis g ade, 2 mL) o 20 min in a pep ide syn hesis essel p io syn hesis.
Coupling cycle consis ed o he emo al o Fmoc p o ec ing g oup wi h a solu ion o pipe idine in DMF
(20%, 2 mL) o 10 min and hen he mix u e was il e ed and he esin was washed wi h DMF (3 x 2 mL,
1 min). The amino acid coupling was ca ied ou by ea men wi h a solu ion o α-amino acids (4 equi ),
N-HBTU (3.95 equi ) in DMF (2 mL), which was mixed wi h DIEA (0.195 M solu ion in DMF, 1.2
equi ) 1 min be o e he addi ion and he esul ing mix u e was shaken by bubbling A o 15 min. Finally,
he esin was washed wi h DMF (3 x 2 mL, 1 min). The e iciency o each amino acid coupling and
dep o ec ion was moni o ed employing he TNBS es .S3
Fo au oma ed syn hesis, a a ian o he p e ious p o ocol was used ins ead, acco ding o
manu ac u e ’s ecommenda ions. 0.05 mmol o Rink Amide esin was placed in o he pep ide syn hesise
eac ion essel, swollen in DMF, ollowed by cycles o Fmoc clea age wi h pipe idine 20% in DMF,
washings (3 x 5 mL), hen amino acid (5 equi 2 M amino acid solu ion in DMF), DIC (10 equi ) and
Oxyma (10 equi ) we e added in o he eac ion essel and mic owa ed o 5 min unde empe a u e
con ol ollowed by washings (3 x 5 mL). All s eps we e pe o med unde ni ogen a mosphe e. A e he
linea pep ide was inished he esin was ans e ed o a di e en eac ion essel o pe o m he pep ide
modi ica ion manually.
Fluo opho e coupling: he Fmoc-p o ec ing g oup o he ini ial amino acid was emo ed by using a
solu ion o pipe idine in DMF (20%, 4 mL) o 15 min and he esin was washed wi h DMF (3 x 3 mL).
The coupling was ca ied ou by he addi ion o a solu ion o 5-ca boxy e ame hyl hodamine o 5,6-
ca boxy luo escein (1 equi ), HATU (1 equi ), and DIEA (0.195 M, 1 equi ) in DMF (2 mL) and he
mix u e was s i ed by bubbling A o 4 hou s. Finally, he esin was washed wi h DMF (3 x 3 mL) and
DCM (3 x 3 mL).
Ace yla ion in he N- e minus: Once he lineal pep ide was inished he ace yla ion capping o N-
e minal g oup was pe o med by s anda d Fmoc dep o ec ion condi ions (20% pipe idine in DMF (2 x 5
mL, 15 min) ollowed by ea men wi h a solu ion o 2,6-lu idine/ace ic anhyd ide (1:1, 3 mL). The esin
suspension was mechanically shaken o 40 min and washed wi h DMF (3 x 3 mL, 1 min) and DCM (3 x
3 mL, 5 min).
S19
Gene al p o ocol o pep ide clea age and pu i ica ion: Finally, pep ides we e dep o ec ed and
clea ed om he esin by s anda d TFA clea age p ocedu e a by using he TFA/DCM/H2O/TIS
(90:5:2.5:2.5, 3 mL pe 70 mg o esin) o 2 h. Then, he mix u e was il e ed, washed wi h TFA (1 mL)
and he pep ide was p ecipi a ed wi h ice-cold E 2O (50 mL). The p ecipi a e was cen i uged and
dissol ed in H2O (5 mL) and pu i ied by RP-HPLC. Finally, he co esponding ac ions we e lyophilised
o a o d he pu e solid pep ides.
2.3.1. Syn hesis o pep ide MP1
Following he gene al p o ocol o he SPPS, MP1 was ob ained a e RP-HPLC pu i ica ion
[Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5
min), 95:5→5:95 (5→35 min)] wi h an o e all yield o 30%. R 14.7 min [RP-HPLC Agilen SB-C18
column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→21 min)]. MS (ESI, H2O): 1578.0 (6,
[M+H]+), 789.6 (100, [M+2H]2+), 526.9 (71, [M+3H]3+). HRMS (ESI): Calcd o C68H125N26O17 [M+H]+:
1577.9702; ound: 1577.9711. See Figu e S15.
2.3.2. Syn hesis o pep ide CF-MP1
Following he gene al p o ocol o he SPPS ollowed by luo opho e coupling, CF-MP1 was ob ained
a e RP-HPLC pu i ica ion [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1%
TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] wi h an o e all yield o 9%. R 14.9 min [RP-
HPLC Agilen SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS
(ESI, H2O): 947.8 (37, [M+2H]2+), 632.2 (100, [M+3H]3+), 474.4 (27, [M+4H]4+). HRMS (ESI): Calcd
o C87H134N22O22 [M+2H]2+: 947.5100; ound: 947.5078. See Figu e S16.
2.3.3. Syn hesis o pep ide TM-MP1
Following he gene al p o ocol o he SPPS ollowed by luo opho e coupling, TM-MP1 was
ob ained a e RP-HPLC pu i ica ion [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/
CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] wi h an o e all yield o 12%. R
15.2 min [RP-HPLC Agilen SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20
min)]. Syn hesis and cha ac e iza ion had been p e iously desc ibed as P8 in Pazo e al.S4
2.3.4. Syn hesis o pep ide MP2
Following he gene al p o ocol o he SPPS, MP2 was ob ained a e RP-HPLC pu i ica ion
[Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5
min), 95:5→5:95 (5→35 min)] wi h an o e all yield o 10%. R 14.4 min [RP-HPLC Agilen SB-C18
column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1578.9 (5,
[M+H]+), 789.6 (100, [M+2H]2+), 526.9 (77, [M+3H]3+). HRMS (ESI): Calcd o C68H124N26O17
[M+2H]2+: 1577.9716; ound: 1577.9711. See Figu e S17.
2.3.5. Syn hesis o pep ide P3
Following he gene al p o ocol o he SPPS, P3 was ob ained a e RP-HPLC pu i ica ion
[Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5
min), 95:5→5:95 (5→35 min)] wi h an o e all yield o 16%. R 15.4 min [RP-HPLC Agilen SB-C18
column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1081.1 (33,
[M+2H]2+), 721.1 (100, [M+3H]3+), 541.1 (39, [M+4H]4+). HRMS (ESI): Calcd o C92H168N36O24
[M+2H]2+: 1080.6515; ound: 1080.6511. See Figu e S18.
2.3.6. Syn hesis o pep ide A g8
Following he gene al p o ocol o he SPPS, A g8 was ob ained a e RP-HPLC pu i ica ion
[Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5
min), 95:5→5:95 (5→35 min)] wi h an o e all yield o 58%. R 9.5 min [RP-HPLC Agilen SB-C18
column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 882.9 (11,
[M+2H+4TFA]2+), 826.2 (22, [M+2H+3TFA]2+), 769.0 (26, [M+2H+2TFA]2+), 712.3 (17,
[M+2H+TFA]2+), 589.2 (30.3, [M+3H+4TFA]3+), 551.2 (78, [M+3H+3TFA]3+), 513.1 (100,
S20
[M+3H+2TFA]3+), 475.1 (77, [M+3H+TFA]3+), 437.1 (48, [M+3H+TFA]3+). HRMS (ESI): Calcd o
C50H103N33O9 [M+2H]2+: 654.9304; ound: 654.9303. See Figu e S19.
2.3.7. Syn hesis o pep ide GALA
Following he gene al p o ocol o he SPPS, GALA was ob ained a e RP-HPLC pu i ica ion
[Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5
min), 95:5→5:95 (5→35 min)] wi h an o e all yield o 0.5%. R 20.6 min [RP-HPLC Agilen SB-C18
column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)]. MS (ESI, H2O): 1528.8 (46,
[M+2H-OH]2+), 1537.2 (17, [M+2H]2+), 1025.3 (100, [M+3H]3+), 769.2 (16, [M+4H]4+). HRMS (ESI):
Calcd o C138H220N34O45 [M+2H]2+: 1536.7981; ound: 1536.7980. See Figu e S20.
2.3.8. Syn hesis o pep ide L17E
L17E was syn hesized and pu i ied as p e iously desc ibed,S5 wi h an o e all yield o 23.3%. R 10.6 min
[RP-HPLC Agilen SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)].
MS (ESI, H2O): 1430.2 (23, [M+2H]2+), 953.9 (100, [M+3H]3+), 715.8 (26, [M+4H]4+), 572.95 (7,
[M+5H]5+). See Figu e S21.
2.3.9. Syn hesis o Pep-1
Pep-1 was syn hesized and pu i ied as p e iously desc ibed,S6 wi h an o e all yield o 3.5%. R 11.6 min
[RP-HPLC Agilen SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)].
MS (ESI, H2O): 1424.2 (15, [M+2H]2+), 949.9 (100, [M+3H]3+), 712.7 (20, [M+4H]4+), 570.8 (13,
[M+5H]5+). See Figu e S22.
2.3.10. Syn hesis o d TAT
d TAT was syn hesized and pu i ied as p e iously desc ibed,S7 wi h an o e all yield o 8%. R 11.5 min
[RP-HPLC Agilen SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→20 min)].
MS (ESI, H2O): 1588.0 (10, [M+3H+6TFA]3+) 1550.2 (10, [M+3H+5TFA]3+), 1162.9 (75,
[M+4H+5TFA]4+), 1134.4 (100, [M+4H+4TFA]4+), 1105.8 (61, [M+4H+3TFA]4+) 884.9 (72,
[M+5H+3TFA]5+), 862.1 (55, [M+5H+2TFA]5+), 699.5 (50, [M+6H+TFA]6+), 680.75 (45, [M+6H]6+),
599.75 (16, [M+7H+TFA]7+). See Figu e S23.
2.4. Liposome p epa a ion
The p ocedu e o liposome p epa a ion has been p e iously epo ed.S8 B ie ly, a hin lipid ilm was
p epa ed using 1 mL o 25 mg/mL o lipid in chlo o o m and e apo a ing he chlo o o m on a o a y
e apo a o and hen in acuo o e nigh . The lipids we e esuspended wi h one o he in a esicula
solu ions desc ibed below o 45 min. The esul ing esicles we e ex uded 11 imes h ough a 0.1 µm
Nucleopo e polyca bona e il e . Ex a esicula componen s we e emo ed by desal ing column
(Sephadex G-50) p e-equilib a ed wi h ex a esicula bu e . The inal concen a ion o esicles is 5 mM.
In he case o ANTS/DPX – encapsula ed LUVs (LUVsÌANTS/DPX) he ex a esicula bu e is
composed by 10 mM MES and 150 mM NaCl, a pH 7.4 o 5.5, and he in a esicula bu e is composed
by 10 mM MES, 70 mM NaCl, 12.5mM ANTS and 45 mM DPX a pH 7.5 o 5.5 espec i ely. The lipid
composi ion is 100% POPC o POPC/POPG a 3:1 mola a io.
In o de o make NBD-labelled lipid esicles, lipids labelled in he pola head (PE-NBD) o in he
hyd ophobic ail (NBD-PE) a e added o he lipid mix u e as 0.5%. The emaining lipid composi ion is
99.5% POPC o POPC/POPG mola a io 3:1. The in a and ex a esicula bu e a e he same (10 mM
MES and 150 mM NaCl, a pH 7.5 o 5.5). In his case, he desal ing column (Sephadex G-50) s ep is no
necessa y.S9
In he case o non- luo escen LUVs, he in a and ex a esicula bu e s a e composed by 10 mM
MES and 150 mM NaCl, a pH 7.5 o 5.5 espec i ely, and he lipid composi ion is 100% POPC o
POPC/POPG mola a io 3:1. In his case, he desal ing column (Sephadex G-50) s ep is omi ed.
S21
Dex an loaded liposomes (LUVsÌDex an) we e p epa ed ollowing he p o ocol desc ibed by Sani
e al.S10 Fi s , he ilm lipid was o med wi h POPC o POPC/POPG 3:1 by o a y e apo a ion and hen in
acuo o e nigh . Thus, he lipid was ehyd a ed wi h a solu ion o 20 mM o 10 kDa FITC-dex an and 20
mM o 40 kDa TM-Dex an, 10 mM MES, 5 mM NaCl a wo di e en pH (7.5 and 5.5). A e ha , he
esul ing mix u e was ex uded 11 imes h ough a 0.2 µm Nucleopo e polyca bona e il e . In o de o
emo e he ex a esicula componen s, he samples we e cen i uged h ee imes o 40 min, 1500 pm a
10 °C. A e each cen i uga ion he supe na an was emo ed and eplaced by esh ex a esicula bu e
(10 mM MES, 7 mM NaCl) a he co esponding pH.
2.5. ANTS/DPX assay
LUVsÌANTS/DPX s ock solu ions (50 µL) we e dilu ed wi h a bu e (10 mM MES and 150 mM
NaCl, a pH 7.4 and 5.5), placed in a he mos a ed luo escence cu e e (25 ºC) and gen ly s i ed ( o al
olume ~2000 µL; inal lipid concen a ion ~125 µM). ANTS e lux was moni o ed a λem 510 nm (λex
353 nm) as a unc ion o ime a e addi ion o MP1 a = 50 s and aqueous T i on X-100 (1.2%, 20 µL,
185 µM inal concen a ion) a = 225 s; each measu emen was made in iplica e. Fluo escence
in ensi ies we e no malized o ac ional emission in ensi y I( ) using equa ion (S1).
I( ) = (I – I0) / (I∞ – I0) (S1)
whe e I0 is he ini ial in ensi y o he esicles alone, I∞ = I a sa u a ion a e lysis. E ec i e
concen a ion o pep ides (EC50) and Hill coe icien (n) we e de e mined by plo ing he ac ional
ac i i y Y (= I( ) a sa u a ion jus be o e lysis, = ~200 s) as a unc ion o MP1 cPep ide and i ing hem o
he Hill equa ion (S2).
Y = Y0 + (YMAX – Y0) / {1 + (EC50 / cPep ide)n} (S2)
whe e Y0 is Y be o e he addi ion o he pep ide, YMAX is Y wi h an excess o pep ide a sa u a ion.
Fo e e sible po e ga ing expe imen s, LUVsÌANTS/DPX (3:1 mola a io POPC:POPG) s ock
solu ions (50 µL) we e dilu ed wi h a bu e (10 mM MES and 150 mM NaCl, a pH 7.4 and 5.5) and
placed in a he mos a ed luo escence cu e e (25 ˚C) and gen ly s i ed ( o al olume ~2000 µL; inal
lipid concen a ion ~125 µM). ANTS e lux was moni o ed a λem 510 nm (λex 353 nm) as a unc ion o
ime a e addi ion o MP1 (10 µM) a = 25 s pep ide addi ion, = 50 s lipid addi ion (POPG, 50 µM),
= 150 s pep ide addi ion, = 175 s lipid addi ion, = 275 s pep ide addi ion, = 300 s lipid addi ion.
Finally, an aqueous T i on X-100 solu ion (1.2%, 20 µL, 185 µM inal concen a ion) was added a =
475 s.
2.6. Dex an elease in esicles
LUVsÌDex an s ock solu ions (25 µL) we e dilu ed wi h a bu e (10 mM MES and 7 mM NaCl, a
ei he pH 7.4 o 5.5) and placed in an eppendo . Then he minimal pep ide MP1 was added a di e en
concen a ions and s i ed o 30 min ( o al olume ~1000 µL; inal lipid concen a ion ~125 µM). The
mix u e was cen i uged o 35 min a 10 ºC and 1500 pm. The supe na an was collec ed and measu ed
by spec o luo ome y: FITC-dex an (10 kDa) was moni o ed a λem 500 - 650 nm (λex 492 nm), TM-
dex an (40 kDa) was moni o ed a λem 560 - 700 nm (λex 555 nm). Fluo escence in ensi ies we e
no malized o ac ional emission in ensi y I( ) using equa ion (S1)
I( ) = (I – I0) / (I∞ – I0) (S1)
S22
whe e I0 is I o esicles alone, I∞ = I a sa u a ion a e lysis. E ec i e concen a ion o pep ides
(EC50) and Hill coe icien (n) we e de e mined by plo ing he ac ional ac i i y Y (= I( ) a sa u a ion
jus be o e lysis) as a unc ion o pep ide concen a ion cPep ide and i ing hem o he Hill equa ion (S2).
Y = Y0 + (YMAX – Y0) / {1 + (EC50 / cPep ide)n} (S2)
whe e Y0 is Y wi hou pep ide, YMAX is Y wi h an excess o MP1 a sa u a ion.
2.7. Ci cula Dich oism
Ci cula dich oism measu emen s we e ca ied ou wi h he ollowing se ings: acquisi ion ange: 300-
190 nm; bandwid h: 1.0 nm; accumula ion: 3 scans; da a pi ch: 1 nm; CD scale 200 mdeg/1.0 dOD; D.I.T.
(Da a In eg a ion Time): 1 s; scanning mode: con inuous; scanning speed: 200 nm/min. Measu emen s
we e done a 10 ºC and 40 ºC in a qua z cell o 0.2 cm pa h leng h a a inal olume o 0.5 mL (bu e 10
mM MES, 150 mM NaCl a wo pH: 7.4 and 5.5) wi h a inal pep ide concen a ion o 100 µM. The
esul s a e exp essed as he mean esidue mola ellip ici y [θ]MR wi h uni s o deg ees·cm2·dmol-1 and
calcula ed using he equa ion S3. The pe cen age o helici y was calcula ed using he equa ion S4.S11 As
his alue is used o compa isons among simila pep ides in di e en en i onmen s, no co ec ion o he
numbe o a oma ic aminoacids is equi ed. Fo he measu emen s in p esence o non- luo escen LUVs
(100% POPC o POPC/POPG a mola a io 3:1), samples we e p epa ed by mixing he pep ides a 100
µM and di e en amoun s o liposomes a di e en lipid:pep ide a ios (1.25, 5, 12, 50). Fo each sample,
a blank measu emen was done p io addi ion o he pep ide, and sub ac ed o he o he measu emen s
be o e analysis.
[Ɵ]MR
=!.#$%$Ɵ
'·$)·$*+,-./$01$/.234+.2
(deg · cm2 · dmol -1) (S3)
Equa ion S3: Fo mula o calcula e he ellip ici y. Ɵ is he ellip ici y (mdeg), C is he pep ide
concen a ion (M) and l is he cell pa h leng h (cm).
[Ɵ]%
=$
|
Ɵ678!!!
|
$9$:;<!
;!;!!
x
100 (S4)
Equa ion S4: Fo mula o calcula e he pe cen o helici y in which he mola ellip ici y a 222 nm is
an absolu e alue.S11
2.8. DLS expe imen s
Non- luo escen LUVs (50 µL, 125 µM) (POPC o POPC/POPG 3:1) we e added o a bu e solu ion (10
mM MES and 150 mM NaCl, a pH 7.5 and 5.5) o a inal olume o 200 µL (20 ⁰C, du a ion = 70 s).
The bes pa ame e s we e ound using he au oma ic mode, a e which he a enua o index was ixed a
6. Measu emen s we e done a e he addi ion o inc easing amoun s o he pep ide (50, 100, 200, 400,
800 µM) o he sample, and ended a e esicle lysis wi h T i on X-100, when he equipmen could no
measu e, as he coun ed pho ons a e 0. The s anda d de ia ion is gi en by equa ion S5.S12
PdI
=
$
%$=>
6.?*
&
:
(S5)
Equa ion S5: Fo mula o calcula e he s anda d de ia ion using he mean and polydispe si y index
(PdI).
2.9. FRET and luo esence expe imen s
The minimal pep ide MP1 was modi ied a he N- e minus wi h ei he 5-TAMRA o 5-
ca boxy luo escein, which cons i u e a FRET pai , o achie e he pep ides TM-MP1 and CF-MP1
espec i ely. The emission o he dono ca boxy luo escein (a λex = 494 nm, λem = 518 nm) was
measu ed in a inal olume o 2 mL con aining a mix u e o 50 µL, 125 µM esicles and 2.5 µM CF-
MP1. The pep ide FRET pa ne TM-MP1 2.5 µM (5µM inal concen a ion o bo h CF-MP1 and TM-
S23
MP1) was added a = 25 s and he luo escence moni o ed un il = 200 s. CF-MP1 emission was
de e mined a = 10 s and = 150 s and FRET e iciency calcula ed acco ding equa ion S6.
E = 1 – (FDA/FD) (S6)
Equa ion S6: Fo mula o calcula e he E iciency (E), FDA is he luo escence o he dono in he
p esence o he accep o ; FD luo escence o he dono alone.
The second FRET expe imen was pe o med using he same pep ide labelled a he N- e minus wi h
TAMRA (TM-MP1). Anionic and neu al esicles con aining 0.5% NBD-PE o PE-NBD we e dilu ed
(50 µL in 2 mL) in MES bu e a wo di e en pH (7.5 and 5.5). The emission o he dono NBD (λex =
470 nm , λem = 531 nm) was measu ed a = 0 s and a e he addi ion o pep ide accep o pai (0.05, 0.1,
0.5, 1 µM) om = 25 s un il = 200 s. NBD emission alues a = 10 s and = 150 s we e used o
FRET e iciency calcula ion using equa ion S6.
Emission luo escence spec a was also measu ed a di e en concen a ions o CF-MP1 (λex = 494
nm) in aqueous solu ion (MES bu e , pH 7.5). Quenching e ec a inc easing concen a ions sugges s a
micelle-like agg ega ion a concen a ions o e 1.5µM.S13 The shi o he peak o maximum luo escence
in ensi y indica es a comple e aqueous sol a ing e ec in pep ide molecules a concen a ion lowe han
1.5µM.S14
2.10. Compu a ional me hods
Since i is gene ally belie ed ha ea ly endosomes sha e he same o e all lipid composi ion as he
plasma memb ane, as hey a e bo h pa o he same ecycling e i o y,S15 his s udy used an asymme ic
human e y h ocy e memb ane model, ollowing he s udy o Tian e al.S16, which in ol es h ee lipid
ypes, including dipalmi oyl phospha idylcholine (DPPC), dipalmi oyl phospha idyle hanolamine
(DPPE), and dipalmi oyl phospha idylse ine (DPPS). In he ou e lea le o he memb ane, he lipid a io
is 9:1 o DPPC and DPPE; in he inne memb ane lea le , he lipid a io is 3:5:2 o DPPC, DPPE, and
DPPS. Mapping o lipid molecules was based on he Ma ini CG o ce ield (Ma ini_ 2.2).S11,S12 Choline
g oups o lipids a e ep esen ed by he CG beads Qo, Qd, and P5 o DPPC, DPPE, and DPPS,
espec i ely. The phospha e g oups o all lipids a e ep esen ed by Qa. The glyce ol g oups and ca bon
ails o all lipids a e ep esen ed by Na and C1, espec i ely.
The pep ide MP1 model was buil ollowing he ex ended Ma ini o ce ield o p o eins using he
ma ine.py sc ip .S11,S13 Pep ide MP1 con ains 3 Leu esidues, 3 A g esidues and 10 Ala esidues, so ha
he o al cha ge o each uni is +3. The e minal edges o he pep ides we e no cha ged.
A andom dis ibu ion o 31 uni s o pep ide MP1 in e ac ion o an asymme ic memb ane composed
by 1309 CG lipids was in es iga ed, wi h an ini ial size o he box o 20 x 20 x 15 nm. A e a sho
equilib a ion, long MD simula ions o 1000 ns o each case we e pe o med in absence and p esence o
an ex e nal elec ic ield o 0.03 V/nm applied ac oss he bilaye . The sys em empe a u e was kep a
310 K using a V- escale he mos a ,S20 and pe iodic bounda y condi ions we e used in all simula ions. The
Pa inello-Rahman ba os a S21 wi h semiso opic p essu e was applied. The an de Waals in e ac ion
cu o was 1.1 nm. In he case o a omis ic esolu ion, AT-MD simula ions we e ca ied ou using he
G omos54a7 o ce ield.S22 The edges o pep ide MP1 we e modelized as ACE and NH2, espec i ely.
The box was sol a ed wi h wa e o TFE, espec i ely. The sys em empe a u e was kep a 310 K using a
V- escale he mos a ,S20 and pe iodic bounda y condi ions we e used in all simula ions. The Pa inello-
Rahman ba os a S21 wi h iso opic p essu e was applied. The an de Waals in e ac ion cu o was 1.0 nm.
Pa icle mesh Ewald summa ion (PME) me hodS23 was used o de e mine elec os a ic in e ac ions. All
simula ions we e pe o med by he GROMACS 4.5.4 packageS24 and esul s we e ep esen ed by Visual
Molecula Dynamics (VMD) 1.9 so wa e.S25
2.11. Plana lipid bilaye eco dings
Expe imen s we e pe o med in plana lipid bilaye memb anes (BLMs) o med by he Mon al and Rudin
echnique.S26 The eco dings we e ca ied ou by using bilaye s o 1,2-diphy anoyl-sn-glyce o-3-
phosphocholine (DPhPC) ac oss a 80-100 µm diame e hole o a 25 µm hick PTFE (Good ellow
Co po a ion, Mal e n, PA, USA). Bilaye s we e o med by i s p e ea ing he ape u e wi h a d op o
1% ( / ) hexadecane in n-pen ane. The bu e solu ion (0.5 M o KCl and 10 mM o MOPS) was added
o bo h 1 mL chambe s, hen, DPhPC in n-pen ane (5 µL, 5 mg/mL) was added o bo h wells; a e ha ,
he sol en e apo a ed. A bilaye was o med when he elec oly e was lowe ed and aised, b inging he
S24
wo lipid su ace monolaye s oge he a he ape u e. The capaci ance o he bilaye was checked and he
s abili y o he bilaye was moni o ed o 5 min a +200 mV, obse ing no luc ua ions in he cu en .
A e plana memb ane o ma ion he pep ide dissol ed in MQ wa e was added o bo h chambe s a 25
µM. The whole BLM conduc ance was measu ed a ol age-clamp-mode-holding po en ial om ans o
cis chambe . Raw ion cu en eco ding was acqui ed a 1 kHz a a sampling equence o 5 kHz. The
cu en was ampli ied by using an Axopa ch 200B ampli ie (Molecula De ices), digi ized wi h a
Digida a 1440 A (Molecula De ices) con e e , while da a collec ion was done by he Clampex 10.7
so wa e (Molecula De ices). Da a p ocessing and analysis was ca ied ou wi h Clamp i 10.7 ( he
esul ing cu en was il e ed using Low pass Bessel (8-pole)). All Axons ins umen s and so wa e a e
p oduc s o Molecula De ices (Sunny ale, CA). Acco ding o he po e conduc ance, he pep ide po e
size was calcula ed ollowing he Hille equa ion (S7).S27 The conduc i i y o 0.5 M KCl 10 mM MOPS a
25 °C is 5.1 S·m-1, he adius and l he leng h o he po e (4.7 nm) ( heo e ical hickness o DPhPC
memb ane). F om he single channel elec ical eco dings, he cu en o he open s a e is a ound 1,100
pA and he es ima ed diame e is a ound 3.16 nm; o long opened s a es is a ound 1,900 pA and he
diame e a ound 4.4 nm.
1
(=%
$
) +%+,
2
&
·%
$
/
+,:
&
Equa ion S7. Hille equa ion. g is he po e conduc ance; l he leng h o he po e, ρ he esis i i y o he
eco ding solu ion and he inne adius o he po e.
2.12. Cell cul u e expe imen s
2.12.1. Cell lines and cul u e
HeLa, Ve o, and A549 cells we e g own in he same condi ions: a 37 ºC, 5% CO2, in Dulbecco’s
Modi ied Eagle’s Medium (DMEM; 4500 mg/L glucose, L-glu amine, sodium py u a e and sodium
bica bona e), supplemen ed wi h 10% e al bo ine se um and 1% o Penicillin-S ep omycin-Glu amine
Mix.
Human e inal pigmen a y epi helium ARPE-19 cell line was main ained on Dulbecco's Modi ied
Eagle's Medium/Nu ien F-12 Ham (4500 mg/L glucose, L-glu amine, sodium py u a e and sodium
bica bona e) supplemen ed wi h 10% FBS and 1% Penicillin-S ep omycin-Glu amine Mix (Fishe ) a 37
ºC, 5% CO2 in an INCO 108 incuba o (Memme ).
To gene a e a C e Repo e -exp essing HeLa cell line, HeLa cells we e ans ec ed wi h he plasmid
C e Repo e (a gi om Niels Geijsen; Addgene plasmid # 62732).S28 In his plasmid, he gene o
dsRED is lanked by wo loxP si es and ollowed by EGFP gene. In he absence o ecombina ion, he
s op codon om dsRED p e en s EGFP ansla ion, while C e-media ed ecombina ion emo es he
dsRED gene, swi ching he exp ession om he ed luo escen p o ein o he g een luo escen p o ein.
S able ans ec an s we e selec ed by main aining he cell cul u e in p esence o 0.5 µg/mL pu omycin
(Enzo Li e Sciences).
2.12.2. Cellula up ake and endosomal elease o dex an
Fo li e cell imaging, cells g own on glass bo om dishes we e washed wi h DMEM s ock and
incuba ed o 1 h wi h a solu ion o pep ide and dex an, using di e en concen a ions o MP1 and 0.25
mg/mL 10 kDa Alexa488-Dex an, 0.25 mg/mL 10 kDa TM-Dex an, 1 mg/mL o 40 kDa FITC-Dex an,
o 0.25 mg/mL o 70 kDa FITC-Dex an. In co-incuba ion expe imen s, 0.25 mg/mL 10 kDa TM-
Dex an and 1.25 mg/mL 70 kDa FITC-Dex an we e used. A e ha , cells we e washed wi h DMEM
wice and incuba ed o 3 h wi h DMEM supplemen ed wi h 10% FBS. Finally, cells we e washed wi h
DMEM wi hou phenol ed and obse ed wi h a spinning disk con ocal mic oscope. Fo lysosomal
labelling, cells we e incuba ed o 30 min wi h 100 nM o LysoT acke Deep Red a e he 3 h incuba ion,
and washed be o e imaging.
Fo he expe imen s in he p esence o endocy osis inhibi o s, HeLa cells we e seeded he day be o e
in 8-well mic o-slides (ibiT ea , Ibidi) a 30000 cells pe well, washed and ea ed wi h chlo p omazine
(30 µM), dynaso e (80 µM), me hyl-β-cyclodex in (5 mM) o EIPA (50 µM) dilu ed in DMEM wi hou
(S7)
