Chem.Comm. 1

Materialsandmethods

Materials.1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC),brainandeggsphingomyelin(SM)and

egg phosphatidylcholine (PC) were purchased from Avanti Polar Lipids. Cholesterol (Chol) was

purchasedfromSigmaAldrich.Twobuffersolutionswerefreshlypreparedandusedas follows i)a

fusionBufferAcontaining10mMTris,150mM,NaCl3mMandCaCl2andii)animagingBufferBof

10mMTris,150mMNaClatpH7.4.Buffersolutionswerefilteredbeforeusewitha0.2μmporesize

inorganicmembranefilter.

Preparationofsupportedlipidbilayers.DOPC,SMandCholweredissolvedinchloroformtogivea

final lipid concentration of 10 mM. Aliquots of DOPC and SM or Chol solutions were mixed in

differentDOPC:SMandDOPC:Cholmolarratios(either70:30or50:50),pouredintoaglassvialand

evaporatedtodrynessunderanitrogenflow.Multilamellarvesicleswereobtainedbyhydrationwith

theBufferAsolutiontogiveafinallipidconcentrationof1mMandthensubjectingthevialto3x2.5

mincyclesoftipsonicationtoobtainunilamellarvesicles.

CircularmicasurfaceswereusedassubstratesforAFMexperiments.Priortouse,micasurfaceswere

glued onto Teflon discs with epoxy-based mounting glue. Phospholipid supported bilayers were

prepared by the deposition of the small unilamellar vesicles suspension on freshly cleaved mica

followed by incubation at 59°C. The samples were then slowly cooled to room temperature and

thoroughlyrinsedwithBufferBsolution.

Toxin production, purification and validation. The SM-specific toxin fragment, non-toxic (NT)

lysenin, was expressed in E. coli BL21 (DE3) as a fusion protein with a 6xHis-tag followed by the

fluorescentproteinmCherry(totalMW~45kDa)atN-terminal,aspurifiedandvalidatedpreviously1.

Thechol-specifictoxinfragment, i.e.thefourthdomainofperfringolysine(thetaD4),wasclonedin

pET28 containing 6xHis- and LPETGG-tags in N- and C-terminal, respectively (Fig S1a). It was

generated from pET28/His-mCherry-theta2 by removing the mCherry sequence and adding the

LPETGG-tag.TheresultingplasmidwastransformedinE.coliBL21(DE3)andtheproteinexpressedin

LBmediumfor72hat16°Cafteradditionof0.4mMisopropyl-β-D-thiogalactoside.Bacterialextracts

andproteinpurificationwerepreparedaspreviouslydescribed2.Analysisofthepurifiedproteinby

westernblotrevealedrecombinantthetaattheexpectedsize(~16kDa;Fig.S1b).Themostenriched

fractionswerepooled,concentratedusingVivaspinturbo15columns(Sartorius),andtheimidazole

was removed by desalting onNAP-5 columns (GE Healthcare). Purified proteinwas finally kept in

aliquotsin10mMHEPES(pH7.2)10mMNaClandstoredat-80°Cuntiluse.Proteinconcentrationin

thepurifiedfractionwasestimatedbymeasuringA280andassumingamolarabsorptivityof44500M-

1.cm-1.From1lofculture,weobtained10gofprotein.Thetoxinbindingspecificitywasverifiedon

MLVscontainingincreasingamountsofChol(Fig.S1b)andpreparedaspreviouslydescribed1.

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2018

Chem.Comm. 2

AFMtip functionalization.Lysenin-andθ-functionalizedAFMtipswereobtainedusingNHS-PEG27-

acetal linkers3.ToobtainNHS-PEG27-acetaltips,AFMcantileverswerefirstcleanedwithchloroform

for10min,rinsedwithethanol,N2driedandthencleanedfor15mininanultravioletradiationand

ozonecleaner(UV-O,Jetlight,CA,USA).Thecantileverswereimmersedinanethanolaminesolution

(3.3gethanolaminein6.6mlDMSO)overnightandthenrinsedinDMSO(3x1min)andethanol(3x

1min), followedbyN2drying.To attach the linker to theAFMtip,1mgofNHS-PEG27-acetalwas

dilutedin0.5mlchloroformwith30µltriethylamineandcantileverswereimmersedinthissolution

for 2 h. The cantileverswere then cleaned 3 x 10min in chlorofom and driedwith N2. Next, the

cantileverswereimmersedina1%citricacidsolutionfor10minutesandrinsedwithpurewater(3x

5min),followedbydryingwithN2.

To obtain Lysenin-tips, 100 µl of a 100 µM tris-nitrilotriacetic amine trifluoroacetate (tris-NTA)

solutionwaspipettedontothecantileversand2μlofafreshlyprepared1MNaCNBH3solutionwas

then added and gently mixed. The cantilevers were incubated for 1 h, then 5 μl of a 1 M

ethanolaminesolutionpH8.0wereaddedfor10minutestoquenchthereaction.Cantileverswere

washed in Tris buffer (3 x 5 min) and then incubated for 1.5 h with 100 μl of a 10 μM Lysenin

solution.Lysenin-tipswererinsedwithTrisbufferandstored in individualwellsofamultiwelldish

untilusedinAFMexperiments(typicallywithin48h).

θ-functionalizedAFMtipswereobtainedbyincubatingNHS-PEG27-acetalcantileversin100µlofa1

mMGGGGGGGGGGKpeptide(Gly10Lys,Genscript,USA)solutiontowhich2μlofafreshlyprepared1

MNaCNBH3 solutionwasadded.After1h, then5μlofa1Methanolamine solutionpH8.0were

addedfor10minutestoquenchthereaction.Cantileverswerethenincubatedwith20µlofa10µM

θ-toxinsolutionand20µlofa10µMSortaseAsolutionfor1hat37⁰C.θ-tipswererinsedwithTris

bufferandstoredinindividualwellsofamultiwelldishuntilusedinAFMexperimentsthesameday.

FD-based AFM on supported lipid bilayers. AFM experiments were performed with a Bioscope

Resolve AFM (Bruker) operated in “PeakForce Tapping QNMmode” in imaging Buffer B at room

temperature (≈24⁰C). Rectangular Si3N4 cantilevers (AC40, Bruker) with a sharpened tetrahedral

silicontip,nominalspringconstantsof0.09N/mandresonancefrequencyinliquidof≈25kHzwere

used.The springconstantof thecantileverswascalibratedusing the thermalnoisemethodat the

endofeachexperiment4andwasfoundtobeof0.08±0.01N/m.

InFD-basedAFMmeasurements,theAFMcantileverisoscillatedwellbelowitsresonancefrequency

inasinusoidalmanner,whilethesamplesurfaceiscontouredpixel-by-pixel.Aforce-distancecurveis

recorded for each approach and retraction of the oscillating cantilever. FD-based AFM height,

Young’smodulusandadhesionmapsare thenobtainedbydoingapixel-by-pixel reconstructionof

theacquireddata.FD-basedmultiparametricmapswereacquiredusingaforcesetpointof100-200

Chem.Comm. 3

pN.TheAFMcantileverwasoscillatedverticallyat0.25kHzwithpeak-to-peakoscillationamplitudes

of100nm.Imageswererecordedusingascanrateof0.2Hzand256x256pixels.

Data analysis.Raw FD curveswere processed offline using theNanoScopeAnalysis 1.80 Software

(Bruker).ToreconstructYoung’smodulusmaps,weanalyzedtheapproachpartoftheforce-distance

curves from PeakForce QNMmaps. The best quality of the fit was obtained when by fitting the

contactpartofthecurvewiththeHertzmodel5,6:

𝐹!/! = !!

!(!!!!)

𝑅!/!

𝛿 (1)

whereEistheYoung’smodulus,δistheindentationdepth,νisthePoissonratio,andRisthecontact

radius.We used a Poisson’s ratio value of 0.3. Young’smoduluswas calculated from the slope of

Equation1.

HeightimageswereprocessedusingtheGwyddionfreeSPMsoftware.Afirstorsecondorderplane

fitwasperformed.Imagesdidnotundergofurtherprocessing.

Chem.Comm. 4

Fig.S1.Toxinfragmentproduction(a)andvalidationofthethetatoxinfragment(b).(a)MappingofpET28-expressing lysenin or theta toxin. (b) Theta toxin fragment (16 kDa) binding specificityvalidationonmultilamellarvesicles(MLVs)containingincreasingCholcontents.Thetawasincubatedwith MLVs made of SM and PC and containing (4 last wells) or not (2 first wells) Chol. Aftercentrifugation,pellets(P)containingMLVsandsupernatants(SN)wereanalysedbywesternblottingusinganti-Hisantibodies.The6xHis-taggedtoxinonlybindsChol-containingMLVs(P).

Chem.Comm. 5

Fig. S2. Validating the specificity of the interactions detected by toxin-derivatized AFM tips. Thelysenin-tipwasusedtoprobeaSM-free lipidbilayerofDOPC:Chol (70:30). (a,b,c)Height,adhesionandYoung’smodulus imagesoftheDOPC:Chol(70:30).NospecificunbindingeventwereobservedbetweentheLysenin-tipandtheDOPC:Cholsample. (d,e,f)Aθ-functionalizedAFMtipwasusedtomap a Chol-free lipid bilayer made of DOPC:SM (70:30) (d) High-resolution height image of theDOPC:SM(70:30)bilayerand(e)correspondingadhesionshowingnointeractionbetweenthetipandthesampleand(f)Young’smodulusmap

Chem.Comm. 6

Fig. S3. Mapping SM-enriched domains with bare AFM tips on DOPC:SM 50:50 and 70:30 lipidbilayers. (a,b) Height images of the bilayers and corresponding (c,d) adhesion and (e,f) Young’smodulusmaps.

Chem.Comm. 7

FigureS4.MappingChol-enricheddomainswithaθ-functionalizedAFMtiponaDOPC:Chol(70:30)lipid bilayer. (a) High-resolution height image of the bilayer and (b,c) corresponding adhesion andYoung’smodulusmaps.(d-f)HighermagnificationmapsofsingleChol-enricheddomainsencircledin(a-c) along with the corresponding height, adhesion force and Young’s modulus (E) cross-sectionprofiles.

Chem.Comm. 8

FigS5.MappingChol-enricheddomainswithaθ-functionalizedAFMtiponaDOPC:Chol(70:30)lipidbilayer.(a)High-resolutionheightimageofthebilayerand(b,c)correspondingadhesionandYoung’smodulusmaps.

Chem.Comm. 9

Fig. S6.Mapping Chol-enriched domainswith a bare AFM tip onDOPC:Chol 50:50 and 70:30 lipidbilayers.(a,b,c)Heightimagesofthebilayersandcorresponding(d,e,f)adhesionand(g,h,i)Young’smodulusmaps.

Chem.Comm. 10

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