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PROTEIN ORIGAMI - A PROGRAM FORTHECREATIONOF3DPAPERMODELSOFFOLDEDPEPTIDESMANUAL

ProteinORIGAMIisabrowser-basedwebapplicationthatallowstheusertocreatestraightforward3Dpapermodels of foldedpeptides for research, teaching andpresentations.An aminoacid sequence canbe turnedintoa-helices,b-strandsandrandomcoilsthatcanbeprintedoutandfoldedintoproperlyscaledmodels,withacolourcodedenotingthebiophysicalcharacteristicsofeachaminoacidresidue(hydrophobicity,charge,etc.).Thesemodelsprovideanintuitivevisualandtactileunderstandingofpeptideinteractionswithotherpartners,such as helix-helix assembly, oligomerization, membrane binding, or pore formation. Helices can also bedisplayedasahelicalwheelorhelicalmesh in2Dgraphics, tobeused inpublicationsorpresentations. Thehighly versatileprogrammeProteinORIGAMI is also suited to create less conventionalheliceswith arbitrarypitch (e.g.310-helix,π-helix,or left-handedhelices).Non-canonicalaminoacids, labelsanddifferent terminalmodificationscanbedefinedanddisplayedatwill,anddifferentprotonationstatescanbeshown.

CITATION

Reißer,S.,S.Prock,H.Heinzmann,A.S.Ulrich,"ProteinORIGAMI-aprogramforthecreationof3Dpeptidepapermodelsoffoldedpeptides",tobepublished

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TABLEOFCONTENTS

Citation..............................................................................................................................1

Access................................................................................................................................3Installationforuseonthecommandline.....................................................................................3

Input..................................................................................................................................4Webinterface..............................................................................................................................4Aminoacidsequence(1)..............................................................................................................4N-terminalmodifications(2)........................................................................................................4C-terminalmodifications(3).........................................................................................................4pH-value(4).................................................................................................................................5Helixpitch(5)...............................................................................................................................5Representation(6).......................................................................................................................53or1lettercode(7)....................................................................................................................5Color/blackandwhite(8)...........................................................................................................5Startindex(9)..............................................................................................................................5Shiftgeometry(10)......................................................................................................................6Name(11)....................................................................................................................................6Non-canonicalaminoacids..........................................................................................................6

Output...............................................................................................................................7Commandlineversion:conversionwithinkscape........................................................................7Creating3Dhelixmodel...............................................................................................................8Creating3dbeta-sheetandrandomcoilmodels..........................................................................9Helicalwheelandhelicalmeshrepresentations.........................................................................10

References.......................................................................................................................11

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ACCESS

ProteinORIGAMIisrecommendedtobeusedviathewebinterfaceonhttp://ibg.kit.edu/protein_origami.

Acommand-lineversionisalsoavailable,installationinstructionsbelow.

INSTALLATIONFORUSEONTHECOMMANDLINE

The python source code for use on the command line can be found on github:https://github.com/sabinereisser/protein_origami.Forinstallation,followthesethreesteps:

1) Downloadthepackage

git:

git clone https://github.com/sabinereisser/protein_origami

cd protein_origami

OR

wget

wget https://github.com/sabinereisser/protein_origami

/archive/master.zip

unzip master.zip

cd protein_origami-master

2) Runinstallscriptandsourceexecutable

bash install.sh

. ~/.bashrc

3) Runprogram

protein_ORIGAMI

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INPUT

WEBINTERFACE

Figure1:Web interfacehttp://ibg.kit.edu/protein_origami, subpage "Submit sequence". Fields areexplainedbelow. Hovering over the blue question mark signs shows help information. Fields are explained belowaccordingtothenumbersinthregreycircles.

AMINOACIDSEQUENCE(1)

Typetheone-lettercodeoftheaminoacidsequence.Usecapitalletters(default)forL-form,smalllettersforD-form(CapsLock/Shiftkey).Foruseofnon-canonicalaminoacids,useoneofthelettersB,J,U,XorZ.

Commandlineoption:-s,e.g.-s "MNLVDIAILILKLIVAALQLLDAVLKYLK"

N-TERMINALMODIFICATIONS(2)

ChooseN-terminalmodification:NH3+,formyl(HCO),acyl(R-CO),orNone.DefaultisNH3

+.

Commandlineoption:-nterNH3/FOR/ACY,e.g.-nter FOR

C-TERMINALMODIFICATIONS(3)

ChooseC-terminalmodification:COO- (COO),NH2 (amidated,NHE),NH-R (lipidated, LIP),orNone.Default isCOO-.

Commandlineoption:-cterCOO/NHE/LIP,e.g.-cter NHE

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PH-VALUE(4)

ChoosepHrange:<=7(le7)or>=8(ge8),affectschargesofNH3+terminusandhistidine.pH<=7:NH3

+charge+1,Hischarge+1.pH>=8:NH3

+charge0(becomesNH2),Hischarge0.DefaultpH<=7.

Commandlineoption:-pHle7/ge8,e.g.-pH le7

HELIXPITCH(5)

Chooseangularpitchindegreesforhelixrepresentations.Anyvalueispossible,e.g.α-helix100°,310-helix120°,π-helix≈87°.Defaultvalueis100°.

Commandlineoption:-pitchVALUE,e.g.-pitch 100

REPRESENTATION(6)

Graphicalrepresentationoftheaminoacidsequence.

For 3D paper models, choose "Helix Origami" (helix_origami), "β-sheet" (beta_sheet), or "random coil"(random_coil).Defaultis"HelixOrigami".

For 2D graphics for use in articles or presentations, choose "Helical wheel" (helical_wheel), "Helical meshhorizontal"(helical_mesh_horiz),or"Helicalmeshvertical"(helical_mesh_vert).

Commandlineoption:-represhelix_origami/helical_wheel/helical_mesh_vert/helical_mesh_horiz/beta_sheet/random_coil,e.g.-repres helical_wheel

3OR1LETTERCODE(7)

Showaminoacidnameinstandardoneorthreelettercode.Default:3.

Commandlineoption:-c1/3,e.g.-c 3

COLOR/BLACKANDWHITE(8)

Usecoloredoruncoloredaminoacidrepresentations:colororblack/white.Defaultiscolor.

Colorsare:

• yellow-hydrophobic(Ala,Ile,Leu,Met,Phe,Trp,Tyr,Val)• red-anionic(Asp,Glu,freeC-term)• darkblue-cationic(Arg,Lys,Orn,protonatedHis,freeN-term)• lightblue-polar(Asn,Gln,Ser,Thr,deprotonatedHis)• green-structurallyspecial(Cys,Gly,Pro)

Commandlineoption:-clcolor/bw,e.g.-cl bw

STARTINDEX(9)

Ifthepeptideisafragmentofabiggerprotein,theresiduenumberingcanbeadustedbysettingastartindex.Default1.

Commandlineoption:-bNUMBER,e.g.-b 10

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SHIFTGEOMETRY(10)

In thehelix representations, the startpositioncanbe shifted,e.g. inorder tocentera regionof interest,bysettinganintegervalue.Defaultis0.SeeFigure5.

Commandlineoption:-shNUMBER,e.g.-sh 2

NAME(11)

Theoutputnameofthepeptide.Itcancontainnumbers.

Commandlineoption:-nNAME,e.g.-n mypeptide

NON-CANONICALAMINOACIDS

Ifthesequencecontainsnon-canonicalaminoacids,useB,J,U,XorZinthesequence.If"code"issettothreeletters,thethree-lettercodeisaskedinapop-updialogue.If"color"issettocolor,thecolorisaskedinapop-updialogue,seeFigure2.

Figure2:Dialogueforspecifiactionofnon-canonicalamino-acids.

Onthecommandline,theuserispromptedtoprovideinformationaboutthree-lettercodeandcolor.

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OUTPUT

Output intendend for 3D paper models ("Helix Origami", "β-sheet", or "random coil") is produced in PDFformat."Helicalwheel","Helicalmeshhorizontal",or"Helicalmeshvertical"areadditionallyproducedinEPSandPNGformat.

COMMANDLINEVERSION:CONVERSIONWITHINKSCAPE

ThecommandlineapplicationproducesSVGfiles,whichcanbeconvertedwithinkscape(inkscape.org)toPDForPNG

• viathegraphicalinterface:

File->Saveas(SelectPDF;alwaysfor3Dpapermodels)

File->ExportPNGImage

• viathecommandline:

PDFfromat(seperatelyforeach*_1.svg*_2.svgetc.,whencreating3Dpapermodels):

inkscape name_1.svg -A name_1.pdf

PNGformat

inkscape name.svg -e name.png -D -b white -d 300

-distheoptionfordpi,ifyouwanthigherquality,increase

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CREATING3DHELIXMODEL

Figure3:Assemblyof3DProteinORIGAMImodelofanα-helix,asillustratedherefortheamphipathicpeptideTisB (1).First, thehelixmeshrepresentation isprintedoutonpaper (Lys-26 is zoomed in to illustrate the3-lettercodeandnumbering).Ifthepeptideistoolongtofitononepage,additionalpageshavetobecutandgluedtogether(A).Rollingupthesheet(s)alongthelongaxisandgluing(B),a3Dpeptidemodeliscreated(C).Residues are represented by circles that are color-coded according to the type of side chain: yellow -hydrophobic,red-anionic,darkblue-cationic,lightblue-polar,green-special.

Commandline:protein_ORIGAMI -n TisB -s "MNLVDIAILILKLIVAALQLLDAVLKYLK" -sh 2

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CREATING3DBETA-SHEETANDRANDOMCOILMODELS

Figure4:(A)Buildinga3DProteinORIGAMImodelofaβ-strand,asillustratedherefortheamphiphilicpeptide[KIGAKI]3(2–4).Upongluingandfoldingintoadouble-sidedzig-zagribbon,allhydrophobicsidechainsareseeninyellowononeside(upper3Dmodel),whileallchargedsidechainspointtotheotherfaceintheirrespectivecolor (bottom 3D model). On each face of the extended β-strand, every second “invisible” amino acid isindicatedingrey.Severalsuchβ-strandscanbeassembledside-by-sideintoaβ-sheet(notshown).(B)Randomcoilrepresentationofanintrinsicallydisorderedsequence,asillustratedhereforthecell-penetratingpeptideCLIP6(5). Inthiscase,theflexiblechainofaminoacids isrepresentedincoloronbothsidesoftheextendedpaperstrip.

Command line (A): protein_ORIGAMI -s "KIGAKIKIGAKIKIGAKI" -cter NHE -repres beta_sheet -n KIGAKI

Command line (B): protein_ORIGAMI -s "KVRVRVRVpP TRVRERVK" -cter NHE -repres random_coil -n CLIP6

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HELICALWHEELANDHELICALMESHREPRESENTATIONS

Figure5:2Dgraphicsareusedtoillustratehelicalpeptides,asshownfortheamphipathicpeptideTisB(1).Ile-10 is zoomed in to illustrate thestandard1-lettercodeandnumbering. (A)Helicalwheel representation, (B)helix mesh vertical, (C) helix mesh horizontal. Residues are represented by circles that are color-codedaccordingtothetypeofsidechain:yellow-hydrophobic,red-anionic,darkblue-cationic,lightblue-polar,green-special. (B)and(C)aregeneratedwithshiftgeometry=2 tomovethepolarstrip(red, lightblueanddarkblueaminoacids)intothefigurecenter.

Command line (A): protein_ORIGAMI -n TisB -s "MNLVDIAILILKLIVAALQLLDAVLKYLK" -c 1 -repres helical_wheel

Command line (B): protein_ORIGAMI -n TisB -s "MNLVDIAILILKLIVAALQLLDAVLKYLK" -c 1 -repres helical_mesh_vert -sh 2

Command line (C): protein_ORIGAMI -n TisB -s "MNLVDIAILILKLIVAALQLLDAVLKYLK" -c 1 -repres helical_mesh_horiz -sh 2

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REFERENCES

1. Steinbrecher,T.,S.Prock,J.Reichert,P.Wadhwani,B.Zimpfer,J.Bürck,M.Berditsch,M.Elstner,andA.S. Ulrich. 2012. Peptide-lipid interactions of the stress-response peptide TisB that induces bacterialpersistence.Biophys.J.103:1460–1469.

2. Blazyk,J.,R.Wiegand,J.Klein,J.Hammer,R.M.Epand,R.F.Epand,W.L.Maloy,andU.P.Kari.2001.Anovellinearamphipathicβ-sheetcationicantimicrobialpeptidewithenhancedselectivityforbacteriallipids.J.Biol.Chem.276:27899–906.

3. Lu, J., J.Blazyk,andG.A. Lorigan.2006.Exploringmembraneselectivityof theantimicrobialpeptideKIGAKIusingsolid-stateNMRspectroscopy.Biochim.Biophys.Acta.1758:1303–13.

4. Wadhwani, P., E. Strandberg, N. Heidenreich, J. Bürck, S. Fanghänel, and A.S. Ulrich. 2012. Self-assemblyofflexibleβ-strandsintoimmobileamyloid-likeβ-sheetsinmembranesasrevealedbysolid-state19FNMR.J.Am.Chem.Soc.134:6512–5.

5. Medina, S.H., S.E. Miller, A.I. Keim, A.P. Gorka, M.J. Schnermann, and J.P. Schneider. 2016. Anintrinsicallydisorderedpeptidefacilitatesnon-endosomalcellentry.Angew.ChemieInt.Ed.55:3369–3372.