selecting near native conformations in homology modelling

8/6/2019 Selecting Near Native Conformations in Homology Modelling

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I $+ ,11 r.,-a '':'"lortlP,oteinSciencelg9E,\,7.1?72-l8OCambridge niversity rss. rintedn thUSA'Copyright@ 1998TheProteinSociely

Selecting ear-native onformationsn homologymodeling:The role of molecularmechanicsandsolvation erms

AJTTJANARDHAN ANDSANDOR VAJDADepanmcnlfBiomedic!| ngincering'oston niversity,4cummingtont''Boslon. assachusens2215(RrcErvED ugust6, 1997;ACCEPTEDpril l?' 1998)

AbstractA free energy unction, combining molecular mechanicsencrgywith emPi.ical solvation andentropic tcrms, is used orranking neir-naiive confomations that occur in the conformational searchstepsof homology modcling, i.e., side-chainsearchhd loop closure calculations,Correlations betwcenthe free enetgyatd RMS deviatiol from the X-ray struclurcareestablished. t is shown lhat generally both molecular mechanicsand solvation/entropic terms should be includedin the potential. The identification of near-nativebackboneconformations is accomplishedprimarily by the molecularmechanicserm that becomes he dominant contribution to the free energy f the backbone s even slightly sttained, asf.equcntly occufs n loop clo$urecalculations.Both termsbcomeequally important if a sufficiently accuratebackboneconformation is found. Finalln rhe selectionof the bestsid-chainpositions for a fixed backbone s almost completelygovemed by the solvation term. The discrimimtory po\yer of thc combinedpotential is demonstratedby evaluating theiree energiesof protein models submittedto the first meeting on Critical Assessment f techniques or protein Structo'ePrcdictio; (CASPI), and comparing them to the frce energiesof the native conformations'Keywords: free enerSy: oop closure; protein conformation; side-chainsearch

Identifying native and near-native olds amonga set of conforma-tions is an imponant step in a variety of applications involvingconformationalwarch. Here we focus on homology modeling thataims ro build the smrture of a target prorein beginning with thecoordinatesof a homologue serving as the template. Homologymodeling employs searchalgorithms to determinethe sructure ofcertain loop regions and to place nonconservedside chains. Ac'cordingly, this papr deals with the problems of ranking near-nativc;onformations that are generated by side-chainsearchandloop closurc algorithms, or have beenobtainedfrom a tenplate byva ous homology modeling FoceduresAs shownby Novohy and co-workers Novotny et al ' 1988),molecularmechanics neBy functionsmay be unable o diEtin-suish betwecDcorect and misfolded colfomations' While mo"tcukr mechanicss auseful tool foJstudyinglhe effectsof covalentbonding,excluded olumes,and coulombicetecttostatics,t is in-adeq ale for a thermodynamical description of stable, compactprot;in folds lhat may b heavily influenced by the nature of theiriolvent exposedsr.rrfacesvajda et al', 1997) A further disadvan-tage of molecular mechanics is ils endency to yield a ruggedenergysurfacewith countlessocal minim4 resulting n an et-

tieme sensitivity to small penurbations in the atomic coordinates'Due to thesedifficulties, mol@ular mechanicshas been incteas-ingly replaced by simplified, structure-basdPotentials (Vajdaet al., 199?;sippl, 1995).The main applications avebeen oldrcognition Codziket al., 1992:Maiorov & Crippen,1992;Bry-ant & Lowr"n"", 1993;sippl, 193), andab nitio foldingof poly-pepiides r smallproteins Wilson& Doniach,1989;SrinivasanRose,1995; emigan& Baha!,1996:Yue& Dill' 1996) However'there $ no guaranteehat any of theseempirical Potentialswill beable o distinguish easonably ell between aliveandnear-nativeprotein olds (Huanget al., 1996;Park & l,evitt, 1996)'The goal of this paper is lwofold. First, we dscribe a frceenergypotential that expand$a molecular mechanicsenergy unc-tion by empirical solvationand entropic ems' and is computa-tionally efficient to be used in a variety of applications nvolvingconformatlonal seaich. The same potetrtial has ben extensivlyused or docking and binding free energycalculation (Vajdaet al"1994:Gulukotael al,, 1996:King et al., 1996iWenget al ' 1996)'Although the various tems of the free e[ergy function are basedon very different models, we have show'l that they arc consistentwith eachother and with thermodynamicdata (vajda et al" 1995:weng el al., 1997).However, n dockingand binding ree energycalculation, it is frequently assumedhat either both moleculesaredgid, or the energychangedue to flexibledefomations s smallJative to orhercontributions to the bilding free energy(Novotny

t772RDrinteou$ls o: Sandor ajda Dpafiment f BiomedicalEn8ineer'ing,Iiorton nivcnity.44 ummington l Boston. $sachusens22l5:e-mail: [email protected].


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\onfonnational Irce enerey in homology modelirget al., l9E9; Vajdaet al., 1994; Jackson& Stemberg, 1995; Nau-"t i "i ., ot., t9i5; Verkhivker et al.' 1995; wallqvist et

al'' 1995)'These assumplionsclcarly do not apPly to homology modelingwherethesearchs performedover a setof differnt confomationsthat may be heavily suained. ln fact' as we will show,the intrnalenergy erms that occur dueto thc deformations of the polypeptidcgeorietry aregenerally vry impofiant' andcan evendominate thefree nergyexpression.Our secondgoal is to a"monstrate the usefulnessof the empir-ical frrr energy potential in disringuishing nativc or ncat-natlveDroteinconfotmalions from others that arc less native-like' Testsinvolvc eosemblcsof depoysgeneratedby sealch algorithms thatare part of homology modeling, i.e., side-chain searchand loopcloaure.The discriminatory power of the potential is further dem-onstratedby evaluating the fre energiesof Protein models sub-mitted to th fir$ meetingon critical A$sessment f techniques orDroteil Structurc I\diction (CASPI), and compating them 1o heiree ene4ies of the native conformations.

Empi cal ffte energy utrctionsHere we desciibe the basic Finciples of ftee energyevaluationbyempirical apFoaches, witt| more details given in Methods Thefrec energydifrerelce, AG = G ' C"'vlrrre Go is lhe fre Encrgyin a rfeaence onformation, is calculatedby the expression

r7'13will funher discuss, his approachhas substantialshoncomings nf(eeeDergy alculatior$. I! fact, sinc he solute-solvcnt and solut-solute viw ierms are brsed on very diffetent models, the fre!energy function is very sensilive to small perturbations itr tteatomic coordinaies, leading to a rugged ftee nergy suna@ as mthe case of mole4ular mechanics.

An alternative approach, rcquently used n binding free enerEycalculations, is basedol the approximation that the solutc-soluteand solute-solYenantrfaces are equally well packed, and hencefte van der Waals contacts lost betwen solveni and solute alebalancedby new solute-solute contactsformed upon protein fold-ing (Adamson,1982;Novohy et al.' 1989;Nichollser al" l99l)'Due to this cancellation bodl solute-solvent and solute-solute vander waals ierms can be excluded from the model' Within theframewort of an atomic solvation parametermodel, this can beeasily accomplishedby considering an oryanic liquid as the refer-ence medium, because he ftee energy of ransfer betwen twoliquids inctudes only relatively small, differcntial van der Waalseffects (Weng et al., 1997).The removat of the solute-solute vander Waals term rducesthe molecular mchanicseneryy to

L,E=LE"u"+LEn (2)

AG=AE+ACd-?AS.whcrc E, Ca, and S" denote the moleculat mechanicsenergy, thedesolvation free enefgy, and the conformadonal entropy' respcc-tivly. The energy E is calculaied by a molcular mehanicspo-tential for the conditions of a rcference medium, wbich can beeither vacuum or att organic liquid ln the most general casc, Eincludcs vtn der Waals, eletrostatic, and intemal cnergy termslE = Edw + E"b" + Ei , wherc the intemal (bonded) energyE , i8the sumof bond sttetching, anglebending, tor6ional' and mproper

.tefirs, Eht = Ebad+ End. 1' Eai64o1* Ep*6av-"fltQ desolvatiotrfrec clergy 6d is defined as the free energy of Fansfring theorotein ftom waier into the referenceDedium, and is basedon theclassical aornic solvation paramete. (ASP) model (Eisenberg &Mchchla[ 1986; Wesson& Eisenberg,1992)' The rcferencestateis a folded conformatiotr, ald hence the difference in entropy, ASci$ restricte.d o side chains (Pickett & Stemberg' 193).Notice that using Equation I we calculate the ftee energy dif-frencebetween two $tates ather than ust the energy difference.Indeed, both E and Cd rEpresentan entire nsmbleof equiercr-getic sttucturcs, such as sid{haitr rotarners,rathe. than a singloconformation. Each en$emblehas some confotmatiodal entropy'resuldng in the enaopy change term IAS.. Furtlermore, both Eand Gd are implicitly averagedover an ensembleof water config-urations. n particular, Go,ncludes both thc energyand the entropyof desolvarion.since the solvent is not modeled explicitly, $e calculation ofsolveDt-solutevan der Waals (vdv/) intEractionsrcquires approx-imations.The most 8t?ightrorward strategy s to account for theseitrtenctions in the dcsolvation term ACr', which can be accom-plished by using vacuum as the Eference medium (Wesson &Eisenberg, 992;Abagyan& ToFov,1994;Smith& Honig' 1994;Pellequgr& Chen, 199?). Tte solutc-solute van der waal6 ioter-actions are obtained by the usual 6-12 formula- However' as we

rsulting in a rclaivcly "smoolh" free ene.gy function.the


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-

EP

n 0.2- FMSD 4.{.rrom.)Flg.1.Free rcrgiesAC)of tlcoys enerstedyside-charneatch'

EachCONCENsatch ieldsa ong ist of conformattons'ankdlc"o.aing to thcir CON'CBN nergies, ccountingor the ntemalln".w oi tt e ,ia" "rtuinsand heir nteractions ith therstof the"r"tr"i"

-is;"J"" et al, 1988;Bruccoleri Novorny'1992)''r:-r "J rl" trt" l0o lovest ene8y $tructures ereselgctedor""" A* W-ft normalizatio&Freeenetgies re shownor fewer

i""-t fto""""" becausehe van der Waalsenergies f someJruiiur"t aoufanot u" ."aucedothecommon alue n thevanderliauts oonnuri"ntion. igure alsoshowshe freenergyof thei+av structure f ttreHPRproteinafter200steps f minimizationf"i."L tuitft a do0. Noticethat the RMSDof this conformationilighrly dfiers tromzerodue o the minimization'

Anolysis f decoys bmrned y loopclosuteTheanalvsisxploib he ibraryof decoyshathas een eneraM' i" l. t,t-t, -A "o-tnotkersfot a number f

short ooPs y exten--i"" "r"i"-"lo""r ."arches Moulr & James'

986iFidcliset al"

Flg.3. DesolvationreenergiesAGl) ofdetoysgenmtdy side_chainsearch.

199,1). esultsare shown or loop 132-136 rom the sennepro-uot"" it*u -a loop 120-124

rom the dihydrofolate cductaseilit ni*? tu """tgy evaluation, all conformations taken

from,rt" 4""* filt- \I,".. .ubjected lo 200 stepsof minimization As*,rr * a'"scriu"i i" oiscussion, n evaluatingoop decoys he vanjeiwJ, not atizution can b rcplacedby simple minimizationwith little effect on the results. Figur$ 4-6 show the fiee energyiiii"*""" ai, ti" ,n"lecularmJhanics ncrgyhangc E.ardtrr" "tJtution r."" "nergy ifferenceG2'especiively'or oopilz-iir tot z.gu. te iamequantitiesor loop120-124rom3dfr are shown in Figures ?-9E aluation of predicted structur?'l tom CASPIPrior to the CASPI meeting, a number of resesrchgroups pre-Oi"*a-r*atu,"t fot *ven pioteins plovided as homolo$/ model-i""-"*"o irul".i.*n et;1., 1995) Here we calculste he fre"ti"gyif ;t Prediclion for rhe four proteins

wi$ the highest

s 44"o

o"o&*;'j'-o"et s"d,b"ff

A. Janadhan qnd S. vaida

r 1. 5 2

,z:

iMsD tArlgsltfill

Flg,2. Mole.ular mechanicsnergies (AE) of decoys gcneratedby sidc_chain saIlh.Fi& 4.zsg .

"&

g

Frencrgies (AG) of decoys gneratd or loop 132^h-136^.p of


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do o^

q a'.&J:'"_!on:sd'"-?ff' -o oEF

1775Corrforrnotion^lree energyn homologymodeling

o 0. 5 I 15n sD(.{tr|..d.)Ft& 5. MolcculatmcchanicsnergisA) of decoys cncratcdor loop132^r.-t36^tof 2sga.

numbrof prEdictions submine4 aDdcompare r to fie free encqyof the nativc corfomation. Table I showsthe four targetsafld lhebst emplatesavailable in the Protein Data Bank at the time of theprcdiction contesqalong with relevant data illustEting the degreeof difficulty associatedwith rhe modeling of eachproteil The firstand secondcotumnsgive the target namealld temPlatepdb code'rcspectively, wilh lhe re-solurionof fte crystal sltucture in parED-thesis, The thind and founh columns show the sequerce identityand number of gaps,rcspctivly,betwee[ the t8get andteinPlaleproteins. The remaining columnsshow the spreadofRMSD valuesattaincd by the prcdiclors, afld the mcan RMSD, calculated forboth backboncand all heavy atoms.Tables 2-5 show the rcsults of ftec enetsf c.lculations per-formed on the predictions submitled. Some prcdictions have beenleft out due to enors in sequenccor missing coordinates.For aachprotein we van der Waals normalized the submissions,allorting us

- to rcmove the van dat Waals terms from the poteltial. ln all tableswe list the electrostaticencrgy AE L", the desolvation free encrgy

Flg,6. D.solvotion ftre cnerSies ACd) of decoysSeneratedot loop l32D-| 36aP of 2$ga.

$'qS.eto 0.5 ! 1,5FMSD ArE.irn.l

Flg. ?. FrEeenergiesAc) of decoysScneratdor loop l2ocrv-l24ov of3dft.

AGd, the intemal energy AEn defined as the sum of bood lcngth,bond angle, dihedral, atd improper cne.gy tcrms, aDd hc molec-ular merhanics energy A E = LEw + LE,u*The next column, AG,is deined as the sum of norbonded terms only, LG = LE.h" +AGr, and is included to demonst aic the failutE of a fre eneryyfunction tha: tacks the htemal energy tcrm. The last columns incach table, AC, is thc free encrgy function lhal includes bothbondpdandnonbondedenergyterms,AG = AE;. + LEa- + LGaTbc r9aloDs for omitting the side-chain conformational entsopychange erm, fAS" will be discussed n MethoG.

Discu$slonAnalJsis of decoysobtain d b! side-choin searchThe thredisrioguishableclustersof points in Figure I correspondto thlee separate uns involving the side chaios of rcsidugs l-12,33-45, aod 55-66. The separatecircle with a dot represents he

FIISO l^,'g8lds)Fl& 8, Moleculor mechanics e.Ergies (AE) of dccoys getlentcd for loopl200rr-l24crr of 3df..

Is;aEI!.


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l'176

't}lbirc . Comparorivemodeling catessialyzed" "

A' Jonadhonond5' Vajdaleviated y adding olvation.However't app"s hat tritha fixcdbackbone, ftermajor clashes avebeeneliminaledby energyi-itiirir"ton. tttuU"tt "ritcrioDfor scle4tingativeandnear-native,iJ-"rtnio "onfot "tionsis thesolvation teengrgy' ecausc-thcmolecularmerhanics nergy oesrolFovideanystruc0rralnfor-mation seeFigwe2).Arwlysis f decoys btainedby loopclofirreln looDcatculationsothbackbone ndsidchainsvarywithina.hon raemcnt. s shownn Figure for oop 132-136 f 2sga';;; ";fu a corrclationbetween ree energyard RMSD' andhence ele.ting ow Aeenergystructures De an dentifynt-nu,i"" confo.ti"tiont urnong uchdecoys'As shownn Figure5'drecontributionof themoleaulatmechadcserm s muchmorelt-n-i,f"t i" ,ft" "^e of side-chaincarch n fac! theoverallfrJ energyunctionclosely ollows$e molecularmechanicsn-"*".- i"iio"*A to rhe variationof almost40 kcal/mol in

themiiecularmechanic. nergy,hevariatioon thesolvadon nerSy'shownn Figue 6 is negliSible2'5kcal/rnol)'The cntroprc on-Jioti.n, ra's", it *""-srialler (ootshownseparately) hus' hediscriminanri fte molecularmechanicsnergyThetwocompo-n"to "f ,frit ,t-, i.e.' intemalandelctrostaticnergiea'ehave;;ti;fut to eachothet,with lalgervadationn the ntEmalenergyerm 30 tcat/molvs. 0 kcal/mol)'R;;ion 120-124of 3dfr include$a loop thal is exposdo"ow"ii t " gr"u"t "*bnr thal looP 132-136of 2sga'which

sLselv buriediTterefotewe expexthesolvarion ffec! shownnFi;;9, to contsibutemoreo thevariationn the reeeneryyhani"ii" *"i"* l"*. r"ded.or his oop hemolecularnechanicsandso'lvation nergies re on about hesame cale'althoughhemolecularmechanicserm shown tr Figure8 remainshe moieimportanlof the two contibutions Thefteeenergyuaction s a,.flti""fv gooap..ai"t* of the-RMSD, iththeexceptionf a ow"n""gr "iu-rr", "ntt "a

at 0 7 A. Thesameow energy luster eeni" -u3'o iig.*t 8 and9, arrd husbothcomponentsf the fr"ier* tun-"tiondentify this cluster8senetgeticallymorc avor-^tf"1ft- ,ft" nadue.TireconformatioDsrt this clusterPrimarilyJiffer fromthe n"tive in theorietttation f thi Glu-123 idechain'*ii"t , ,ri tt - n"t g" 8-factorof 94'2, r

essentiallyndefinedn.tfr" i-*v t*"at". is in thecaseof lhe 2sgaoop' tbe-intemalenercvaione"oulabe a goodpredictor f the RMSD'althoughti". iimer"t ut .mab. nariation 9 rcal/mol) than he otalmolec-ularmcchanicserm'-- W"iu"" pi"i.uOy used fteenergy otential hat ncluded ll"i. "i ii*i"" I Lut firc internalercrgy ln

panicular'wecal-

Backbone RMSD All .tom RMSDRangc

| 1. 5

flq.9. Dqsolvationu. cncBicsAC/) fdcoysencratdot oop 20dv_t2lc! of 3dfr,

oioirnize.dX-ray structure f the HPRProtcio ln each uB' allconfomations eneratedre n analow RMSD ange'suggcst[I8inafA6f'fCgf,fs notun dealdeviceor side'chain earchNotice'ioo,"""., tnutonly the loo lowesteneqy structutes avebeenretainedtom eachCONGENcakulation,and hereeDstmanyiunrt"i tru"n t". trtut tave igherRMSD Althoughetainingonlyi00 scucturer ivesa relativolypoorsamplingwithineachut\ thedne! rutrsogtherhowhatselectingower reeenqgystructuEsgenJty yi"iat t*"t RMSD'Funhermore,hemitrimizedX-raysructurchas he owest reenergy.It is well known hatsolvationeffectsarc mponafl to gu16e,iO"-"tuinpfu""."n (Schifferet al , 193: Johnsoo t al ' 1994)'l""ordiog'to the elationshipelweenhemolecul,'

mechanics""*n" -i nr"rSO, rtown

n Figure2' we canevenconcludhat.oiJuruirn."ft-i.t on ts owr is practicallyuselessn thisProb-'i"m, eftfrougltO" nadationn RMSD is limited amonghc l0oio"".t"n"dy .*"*ta" sclectdtom each un' thc valueof ther.i*"i* ,itrt*r"t ""ergy variesby asmuchas l0 kcavmol'Wtrui . """n *o"t", "n"tgiis substantiallyower han hatof lhen4tivo$lructure anbe attained.By corrtrasqhe solvationermiaii "rro*n in nigut 3mnks hegenetatedonformationsLnostidally,bothamong trdevenwithin theclusters'--,li'tfto*n in Fiiurc I' thewell-knownhortcomingf purely.or*ui* .""rt-i"t potentialsNovotny et al ' 1988) anbeal-

Ta.ger(resolution) Templale(rqsolution)No. ofgAPE. RangSequcnce dcntity(%)

'-*ff

EDN(2.2)CRABP (2-7)HPR(2.0)NM23(2.0)

6na (2.0)2hmb 2.1)lpch(1.8)lndl (2.4)

39143.14t.4175

3.1-5.32.0-3.11.0-4.10.4l'.O

4,9-6-42.6-4.4t.74.31.3-3.1

4.62.71, 5t. 2003,42.1

'From drcCASPInlin8.hResolutionndRMSDvaluesotr given m A."res; on rcsoluof 6"quencc liSnlre usingGcc soft$arc


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Cor{ormationatw enew irrhomologymodeli"STablez, Prcdictionsot CMBP I'- 4",..t act A a aE" aet Lc'Abagyan 26.4Moult I 39-4Mouh2 27.5Sali 45.1Vinals -12.4vi.sls 2 6.9Vin.ls 3 64.5Vricnd 52'7Wcber -8.6webcr 2 -r.8

-o2 32.32. t 22.4-3,2 10.5r0.9 53.4-J.d 78.5-0.3 E9.l-9.t l2.E| 1.0 '14.78.8 74.7

59.1 30.011.1 39.150.4 29.655.5 41.941.0 -1.4145,4 63.1f53.6 &.265.5 43.666.1 2.4'to.g 5.0

62.4?1.652.4

r41.6153.371.079.1

'Ener8yvalues elati\ to thalof thenativcstnrcturebElectro6taticnefgl"DesolvattonrceeneigY.dA,E = L,Eb"a+ LE-rk + LEa dd + aEiqq-..LE=LEid+AE,b. .tL6=LE.vc+LCa.Lc = LE.h + A,Etu+LCd.

culdcd the frEencrgiesof protein unfolding aod showd hat thei9sults ale iI good agetncnt ivith the expcrimentally deErminedvalucs (Weng et al., 199?), sugge$tilg lhal both unfolded andnative foldcd protcin confotmatiotts ale free of sigdficant slrainsthat would affect the folding fre ene8y. By contiast' most loopconformations in the deroy library are heavily strainedevetrafterminimization. This should not comg as a surprise sinc tlE localminimization methodsapplied to each oop confomation ftom lbedecoy library sre expctedto find only local ercrgy minim4 andthus ale generally utlable to proced to a fully r9laxed conforma-tion of the moleculc. The fact that we find very intresting anduseful is that thc molccular mechanics energyvaluesat these ocalmioima conrlate with thc RMSD frgm lhe nalive structwe a5shoivn n Figorcs 5 and 8, andhence canbe used o slect he leastdistorted states.

TableS. Predictions or EDMcroup LE"x"h 6Gt" LEi^d aE' Lc'BiosymKochlMoultsd i Isdi 2Srqi tVinalsVinds 2Wber

,f8.854.545.345.651.631.93s.E14.66.2

13.1t6, l2.1A'2. 58.26.46.60.4

50.4 99.224.t 18.746.2 91.527.5 73.129.4 E1.052.3 U.2102.3 138.2f3l. l 145.76l. t 61.3u2.8 145.5

62.4 112.970.6 .747.4 93.653.8 81.354.t E3.5,10.1 92.438.2 1,10,520.9 l52nt2.9 73.933.0 145.9rBnerBy valuca rBlative lo thal of the nttive $ructul.oElccro6tiric cncrgy.cDcsolvation frEc er4y,dLEn= LEoa+ LE-*4 LEdlh"d,ot LEhr-,..'LE = EEr"!+ LEd.r.ILG = LE*" t LGa.tLO = LE.k + LEid+ LGd.

1777Tfrle4. Prediaionsor HPR'ffi--lEJ AGI LEi^,n aE" LG( AG3Abagtan 23.a -3'lBiosyo 8.0 -2,9KoeI 2 n.a 2.6Mosenkis 35.1 -5.7Mouli ?n.l '3.3vried 35.3 3,2Wcber 24.2 4-5

-2.O 21.8 20.1 18.74J 12.1 5.1 9.8t. E y. 9 30.0 31.83.1 30.9 30.4 33.5I 8 36.9 29.4 3l 21.8 21.9 16,E 18.64.0 39.3 3E5 42.4l3.o 31.2 28.8 41.7

rEncrgy values Flativc to thlt of thc nar c *nrcturc.DEleclr$tatic energy."Desolvatioo free clllgY.nL,E6n= h,Er* + 5B** i AE4n74 + LEq'-n,.'LE=6Etu+LE.b. .tLG=LEd,.+LGa.tLc = 6E.h. + LEi,t + LOn.

Theabove bcervationmayalsocxplainwhy thc vandcrWaalrnormalization an b rplacedby simpleminimizationn loopclosu.eptobtems.Recall hat 're iohoduced andc' Waalsnor-malization ecausehe teenqgy functiondoe$ ot nclude dwterms,and hencepackingenors (e.g.,abmic ovdaps) ouldgounnoticed.Howeve!, f there 6 an overlapof backbone tomsorally distortiod n the btckbonegeometrt' he miDimizationwillsubEtantiallyncreastjhe molccularmechanics nergy ermaodthercby lrc fie energy.E atuationof predicted ructaas rom CASPICRABP,EDN, andHPRconstitutemoderalely ifficulthomologymodeling roblms,wi$ seqoenccdentities rcud 40%b$eentemplate rd taryet,while the task of modelingNM23 is trivial'with'll.sEo sequencedentity.Noticc, however,hat theaveragebackboncRMS deviatiotrs nearly de icalfor HPR with41.4%identit, andNM23 (with 773% idc'fityr, while CRABP andEDN predictions ave dcviations hatale nearly wice as laEe'The ra$onor this is that here ar no gapsFesnt tl thealig[-mentsof HPR aM NM23 with their templates,while lhoseof

Table5. Prcdiaionsor NM23'Croup AGa' AEi"d AE. Lct LCtKoehlS6IiVihioenVriendvr'eberWcbe.2

-t6.7-0.940.6| .20. t

-2.4 2693. 1 0. 9-t.5 lr.5-6.4 39.47.3 t8.76-9 n.5

10.2 -19.t 7.80-0 2.2 3.52.t 39.0 50.650.6 4.E 44.317.5 6.1 24.427.6 ?.0 34.5

'Enoryy vaha's Glativc to that of lhc nativc structurc.lElec!.$tatic cnergt,"Desolvadon ftce ercrgy.d6Ea, = AE-a + tE^d, + LEdr^d6t + LEi.,nrt'AE=AEr, ,+ AE, ' . . .tL6-LEa,.+LGatAG = LE*c + LE- + LG*


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r778CRABP and EDN contain sevcral. Thus' lhe backboles of HPRand NM23 ar fully dlinei by their temPlatcs.Furthermore. theoll aroD RMSD botweenHPR and ts teftplate is noticablysmallerthao thoseofCRABP and EDN, even hough all threesharesimilarsequencc dentity with their le$Pective tcmplates (around 40%)'Clo y then, the mo6t impottala step in homology modeliDg i3backbonecoordinatedetermination, as the accuracyof the back-boncootdinates imits the accuracyof sidethain placement'Results n Tables 2-5 show that the empirical fre energyfunc-tion AG defind by Equation I discriminates fte X'ray stsucnrrefrom the corfomations prcdicted by homology modeliDg in allfour prcblems,often by a considerablemargin. As in the case ofloop closure, the molecular mechanicaenergy is gcrcrally muchlaer than the desolvation term and, apait froln a single case,discriminates he native strllctureson its own. The cxception is lhestructurc by Sali in Table 5, which has the same molccular me-chanicsenergyas the X-ray structure f the targel However'wecan accept hat this prediction with the d-catbon RMSD of 0 43 Ais indisinguishable from the native structure, since such RMSDcan be seenbtweentwo X_ray structuresof the sameprotein'Nodce rhat there is a strong corlation beiwen the backboneRMSD and the intemal energy component of the molecular me-chanicsenergy,As we pointed ou! ihe aliglments of CRABP andEDN (Tables2 and 3, tespectively) contained gaps, resulting inhigh deviations between predicted atld actual backbone coordi-naes. For these wo proteins the itrternal energy,AEr,"' ranls thenativefold as he lowcst energy structurc by far' On theotherhand,the alignme s of HPR and NM23 were gap free, and thus thebackbonecoordinatsof thc taryet ate nearly identical to those ofthe template, cading to relativcly small backboneRMSD Tables4and 5, ;f HPR and NM23, re$pectively' reveal that for ihesepro-teins. the intemal energy of the Fedicted structures is aboul thesameand sometimeseven lower than that of the native Since theinternal energy depnds mainly on the backbone, the necessarysimilarity of the backbone stiuctures in urgct and template pto-teins $ggests a simPle test. tf the intemal energy of a Prdictedtargctconformation is much higher thatr the internal energyof theieriplate, thenrheprediction is likely to havc a di$orted backbonc'Sincesuch dislonions may occur due to erotreous aliglment' lhesimDle test can be verY useful'According to our results, the molecular mechanicsenergy di$-criminate$the native $tructuteamong the predictions, and gener-ally dominates the free energy expression unless the backboneconformation is very close to tbc native, suchas in the prcdiclionby Sali in Table 5. It is interestiog that the desolvation terto at-;Db to conDen$ate or large changes n the molecular mechan-ics ierm. For example' hree prediclions n Table 2 (Vinals I'weber I. attd Webt 2) have lo$'er electrostatic energies han thenative. lt is very likely that these models have beenheavily min-imized using a molecular mechanics energy funcliott' afld suchminimizarions frcquently yield very low electsostaticand van derWaalsncrgies.However' as seen n Table 2, the sarn hremod_els have thi highest values of the desolvation frce eneqy AG,t'However, for theVinal I prEdiction the gompcnsation s not stpngenough,andthe corformation is distinguished from the x'ray dueto its much higher intemal eoergY'During the l&st few yeats a large variety of sttucture-basedattdtrydrophitic potential$havc been developedftat do not ircludei,it"rnh o, .ol""ol* .echanics encrgy rcrms (vajda et al ' 1997)'The primary applicarion of these polentials is threading and abinitio simulationof small proteins.Sincethrcadinguses strain-

A Janardhat and S Vaidafre" backbonessuctu.Es observed n ploteins, the poEntials canperfom rcasonablywell. Similarly, in folding sirdies it is mean'irgful to assume hat local staiDs ae removed on a much shortertime scale harl that of the folding itsclf, andhenceone can reltrainconsidedion io terms epresenlingdesolvationand hydrogenbond-ing. By contrast,out results show that in homology modeling theselection of nativ and near_naiiveconfotmations generally re-quires including molecular mechanicsenergy terms in the poten-tial. To emphasiz! this obsrvation, o Tables2-5 we list thc valuesof a nonbonded re. etre.8ypoterdal' 46, that do98not include theintemal encrgy.While Ad was shovn to provide an adequateoolfoi cBlculatittg OF binding ft ercIgy in rereptorligand com-plexes Vajdaet al,, 1994;Culukotaet al., 1996:King et al.' 1996;Weng et al., 1996), in homology modeling ii fails to discriminatethe native structure in two of the four cases(seeTables2' 4)'We haye found lhat for eachhomology modling ta.gl the frercnergiesof the CASPI preiictions were higher than those of theX-ray strudure. However, we were unable to find a conelationbetween free enerSy add RMSD' although such correlationswereeasily identifiable in side-chainsearchand loop closur we ftinklhst comparingentire models built by homology modeling is moredifficult than comparing simple decoys' all generatedby the samernethod.ln fact, homology modling requiresnot only loop closurgandside-chainsarEh, ut alsothe alignmentof target and emplalesequences,he slectionof loop regionsto bebuilt' and :he refine-ment of the derived strucnlres by some typ. of encrgy minimiza-tion. The variou$ groups usedvery diferent assumptionsn thesesteps whcn working on their submissionsto cAsPl, rcsulting inpredictions that differ from each other in a varity of ways' Sinceihe diffcrences affect many valiables, we carl regard thesepredic-tioN aspoints defined in a very high dimensionalconfomationalspace.For each target, the st of submitted ptedictions can bercgardedas a sample of ten or fewer points, which is clearly toosmall for the aoalysis of possible inremctior!$'

ConclusionsIn dre cightics Novotny and cowotken (Novotny et al" 1988)made heprotein comrnutrityaccept hat molecularmechanicsaloneis unabla to distinguish between corecl and misfoldod protinconformations,and one has to addmeasuaes f solvalion andpos-sibly enuopiceffeats. t is ntercstingthat ow we mayhavemovedtooiar away from molecular mechanics During the ast few years'simplified potentials have been increasingly used n protein mod-eling, primarily for rlueading and folding simulations' Thespo"tential; luempt to represent the main f@tots thal are known tocontribute to ihe stability of folded Proicins, i'e ' hydrophobic in-teractions and hydrogen bonding, and genetally do not includeintemalenergy erms(Vajdaet al.. 1997)'The results-of the presentpaper indicate that' apan from side-chain sarchwith a fixed backbone,for ranhng near-nativecon_formations thal occur in homology modeling it is vital to includethe irtemal energy tenn, in addition lo ftee energy conuibutionsreprasentingelecirostatic,solvation' andeotroPiceffccls' While onits o\{n thJ internal energy may not be able to discriminate be-twecn native and non-o4tive folds, its addilion to the other termsdn$tically improves fte discriminatiog powr of the fte energyfunction.Although piotein folding is govemed by hydrophobic inter-actions,htdr;gen bonding, andthe lossof conformational entropy'thcse ouantities ate dwarfed in relation to the intemal energyover


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Codon atiotvl fee e eryy n homolog! wdeli'tglarse ractionsof fie conformationsl pace'n prhciple' onecanff;;;;;;;;i;""w of ttrcmolcculebv relaxinsarl defoF,*:io", "r *" potypeptiae eometry,-n9thenusea

simplifid'"rnprnJ p",""iu.

'x,iwevei' themethodeoutinelyusedn con-iorirr,iJnjr-r"nt"rt""

"r"unableo accomplishhis' 8rd mostua-

i*J"r-""j "p i" r*"1 minima hatatosill in the egionof high'ii"-J.^"tgr" r* example,although lre modelssubminedo"iiit i"".'L- *nned bv theirauthon nd unhcr

mininizedir ut,t t in,"rna "n"tgy dominareshefrc! edcrgyn mostcascs'-' iiil*J *" "tpttasized that thc molecularmechanicserms"-"", u"Itr""l' i""rang near-nadveonformation

equireshe#;i"";";;;;; "neirgvtunction hatalso ncludes olvarionlii ""*pi"'"-t. e, theLginning.of a confotmational

earch'the backboneonformationsreusuallystrained'and he nrcmalJn"rgyaw"trt u otrte. teenergyrmsHowever' electingon'ior."ultion, itrt to* inlemalerrcrgymoveshesearchntotegions"i,rtt "'"nf.-"iona sPacewhcrrhemoleculat

mechanicsndi"soivn ion ,t-t o.t "ti the$ame cale'Finally'aftrabackbonei"ii.-",i"" t accepted'heall-atomRMSDcanb furtherre-iuJ t" n sidc-chain earchgovemedby the desolvldon reJi""* ui-". rrt" i*ponanccof themolecularmechanicsnergyiu.-il""nrry u""n "tphasi?,edin a study ocusingon oopclosureilJ;;;;'& ch"", lb7), butwithout cticins tho ncrasingcleof solvationasbackbonc trainsarcreduceo'MethodsFreeenew calcularionThecnersvchange E in Equation wascalculated singversionis-"ith"Zl$RM. f.t* fiid (Brooks tal ' 1983)with adistance-deoendent ieleaniccofficicntc = 4r' atd nonbondeduloffiii. o"ry p"r"t hydrogenswer uscd To refine the proteinsi"i"* t""i"irrgv *luation (i e" to remove anderwaalsclsshesoi ,ot tuntil ao:io-utionsof gometry), ePerfomedithr2ot)o"or-oi tiniti-,ion using heCHARMn poteotial'o! applicdlE ri i", woottnor-"lizttion pace6xe, in '*hich theminimi-iiiioi nr* *tl"a oo,until thc vanderwaalscnergies f diffetentconformatiorswerewilhio I kcal,/molof eachother'--fftt'Oa-ft"tion f."" eneEyAOd s based nthe atomicsolva'tion DrtametrASP)model

1179

iemDerature-inducedrotcinunfolding(Wenget al l997)'-The;Jil; ;;;;,;nsivelv uscd ot calculatinghe ossof en-;;;i,h" ;d" chains hatbecome artof the recePlor-tigandffiil;;;;;;-*"in associati'on owever'heapproqchi.i"tJ"pplpti " t"t "atculatingtheside-chainntropvifferene

oor"""ri i*o rofa"a.onfofiuations'n fact'atlhoughheside{haincitmpl-rrroufO epena nlyonthebackboneonformation' eusrtt" "iing" in trr. *f""nt exposed laof a sidechaino assessf

it i, a"ir"a andhencehas ts entropy'or becomes uriedartd'*""" 'f''.*" riSr""" side-chain ositionscanvary cvenwiih ailJbJ;;"' ;; teftod hasan nhercnterror' In addition' nftor*fogy rnoa"fing te backboncs f the emplate nd hetarget."rif!it;r*, -i t ence hedifferencen side-chainnlropy s

.i"a"J"lill. rrtit atr"rencemaybecompatablen magnitudei" trtf i"i,".-, ..* ot themethod' trdhencen sorne pplicationsi, tuy t" tor" appropriateo ignore he changen side-chain"n*oi". tn punl*ia' we did not ncludeheconformationaln-*py'"'i-eJ;' ias., when valuatinghe tEtenergy f cASPIsu#issiois,while side-chainnfiopyoss ould calculatedyni'l-ta, of rnor" "*urate methds re4uirin8simulation,ot

ter'ativc dtermination f self-consistentotamerptobaDlllues'

lle,iJr ano*"" in the side-chainntropy t$teenwo similatconformationsoesnor ustify theuseof thesc ompulauonallymordemandingmelhods.DecoySenerulmnAs we described' ide+haindecoysweregercrated y firing $ebackboneoordinats.cmovingall $idechai!$fromcettan sur-i"*-*gi"^ "f ,h" IlPn protein:aod hensearchilgor accePtableriJ"-"i'"li pr"""t"" "sing theALL optionof thcoNcEN pro-in is;."or".i "r ul., 9E8;Bruccolefi Novomy' 992)' hesei**rto i.i tiat-.tt"in de.oys nvolled iagment$-12' 33-45'and55-66 of thc HPRProtein'-ttr" toop decoyswete downloadeduon lnp://pmsarcarh'niit lpbeclpiea$o.rttlt, the Piosttr website'cAsPl pre-dictilns aswell asnativestructurcs reavailableor downloadingon thc web d http://Ptedictiottcenterll'8o'// fiom theProteinStructure ediction Center,Lawrence ivermorNationalLabo-ratory,Livermore, alifomiaTestingnahem,tivemethod fIrce eneq! calculqtionGa= 24ci Throughouthis paperwe usdvan der-waalsnormaliz8tionn

whereAi deooteshe solventaccessibleurfaceateaof $c i$ order o avoia henecd or estimatiog dw interactioDstetweenstomicgroupand i is he.o*rpooo,ng *iiliuim"tr (ei"n- t: pt::il Td dt" *lvent. As mentionedn the ntrcduction'atr*,c&M:L*.1t1-r?:e),ottarnearromJ#-ofi";;;'i;$*;'#*"ili"T:1"f.ffi',:lll':l#Tifl:Ti,"ll,";"i',::%XI5fi jr:i"ffiX ria'Ji"i" "onro-*ionatentropyo6s energJcd.withinhe anevorkf heatomicolvationoram-As. s basednatrempiricalntropy"'iil"r"* a i"'.i,trg, eterisp) moaet,his arbeaccomplishcdsing SPsased

n1993)inwhichfiemaximumconformationalentFPy&ofoachvapol.to.'watertrarrsferfreeenergies(Wesson&Ei$enbrg'19sidechainwascalculatedy tr'" "ratti"a-"x!res"i"ii' : -n x Thenvdw inleraclions rnong

roteinatomsarealso ncludedn>,prln(pr, where , denotesheprobabitif ii the th rotamcr'n the free,energv' ndore calculateibasedon the 'nnard-Jonesthefrec energycalculationwe "..urne rtrit oe-"nti." .ia"-"t ain potential. s;e mentioned,he

shoncomingfthis approachs itse[ttopy s lost, .e,,As. = s., ifthc chanSe .Arin he totalsolvent extremesensitivity to aromic Positioos Nveftheless'we at-accessibleurface r"uor trc ,iae cn"rn " mi." ii* eos6or i" tlmptedro use hi; morcsuaightfotwaldmethod, ndcalculatedstandandside.chainsDrfacealgAl(shrakc&Rupley'1973).other-thefteeeneniesfo'thefourta{getsshowninTable!andthewise heentropyoss 6scaled ccorolngJai"! ls., ."h"." o = peaictions..Frior to energyevaluation,eachconformationwasM,/(0.6Ai).minimizcdfor20osleps.ForEDNandCRABPsomeoftheho-we havesho$l thattheaboveEnropyscaleagrgs ery well mology rodelshave bwervan dcrwaalserptgy h6n henativewith side-chain ntropiesbasedon cat#meric'obsewation of (rcsults lot sho*|r).This is not surprising onsideringhatminl-


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t780mization id vacuum can lead to strucluresmole c4mpact than thenatiye (Vajdact al., 1993). Although the desolvationcnergyrvhichinclud6 solute-solvent vdw interactions attcmpts to countractthc artificislly low vdw energies, ts magnitude is too small forcompensation,and hence we gave up the method in favor of vallder Waalsnormalization.

AclrnovldgmertsWewould ite to $!nk zhipingweng ot technical ssisra&e' ndChadesDclisi. JeanGarnierandCsrlo6Carnachooa nsightfuldiscursions. hcolaborarion ctwetn hc audorsandDr.GarnierwtssuppotcdbyNATOcollabomiiyeRsc0fthCrantCRG950265.This work v9asupported ytbeDonors flhc PFoleum scarch und,dministcrd y h AmericanChemicat ocity, ndby gr3ntDE-F602-96ER62263tomthe Deparrnentof EnerB.ReferenccEAbacysn .Totov M. l99l Biasd robabilityMo . Crdoconformdonnl;r.trJ sndclccrosraticalculdionsorFptidsandpot'ins J MoIaiol23r:983-1002.AdsmsonW. 1982.Phtsicul hemistrJl rrdocts. Nc$'York J Wilv,Brooks R,Bruccolcri E Olafsoo D, States J,Swrmiorlh.nS,Krtplu!M.19E3. harmn:A Fogra for macmmolc.ulstncrgy.minimizaton' nddynamicsdcl|l4ions. ConputChen 4i187'217.Bruccolcri E,HabcrE. Novotny . 1988.Stnctur.of antibody yPcrvarirbleloop6Bproducdy ! confornttional !tchalSoritlun.Natl'/7el'ondon)3$:59-568.Bruccolcri E,Novotny. 1992. ntibodymodclingsing hconform{tionalsrrch lo8lam ongn.t{tvttowtha& I 96-106.BryantSH. iwrcnc. CE. 1991.An cnPincalcnerg/ unctionor thtddingprolrin .qucncehtough hefolding motif.Prdtirr SttuttFutct Gcneit6t92-112.Eiscnberg, Mcl$hlah AD. 19S6Solvadonnergyn proteinoldingandbilld'il|i9Naturelnndon)319: 99-203Fideli,K. stemR BaconD,MouI J. 1994.ConParisonf syslematice3lchalld d{abasenthodsor constNctingsgmntsf prolcinstuclurc.Pto't in Eat 7t95W.CodzikA. KolioskiA, Skolnick . I92. TopologymgerprindngpFoachofie invcrscoldrn!pmblcm. MolAioI227:221-23E.Culukora , VajdaS.Delisi C. 1996.Pcptide ockingsing dynamic o_

'ls'rnnint.r ConputChen 7:4l942aHu|;c E. SubbirhS, Ibri J, lt'itt M 19 Usinga bvdrophobico rcl' ienial to evaluatealivcandncat'nativeoldsSemrrlcdby lnol'lulardymrnics i|nulationi. Mal BioI257716-'125.JrckiotrRM,st mbca MIE 195. A continuuttrmodelor protcin-proternint rActio|t3:pplicationo 6e doclingproblcm., t{ol Biol250,25A-n5.J.miSanRl. Bahar . 196. SEurhne_derivdotontialsndprorcin imula_rion*.Cat OpinStructBiol6:195'm9.Johnson S. Srinivassn , Sowdhamini , BlundellTL 1994.Knowl.ng-bascd rot r modelinS. /iricalR.ltiewsn Biochenitrvoad MolecularRiobsy29\t tl-68.KinsBL.VaidrS, Dc|-iii C 1996Enpiricrl fr.t ener$,asa arg.l uocriofln;oc*ing'ud dsign: pplicationo HIv_l piol[senhibitots.FEB.' zlr3A81-91.MaioovVN, CriPPGnM. 1992.Co rct po&nrialhstEcognizcshcorrcclfoldingof Slobulat ror.itrs. Mol Bbl227f7(-888.MosirnaDn.Mclcshko , IaE.s MNG. 1995.A crilicalassasnctrt f com-paraiiv.moL.ularmodcling fieniarysttucturcsfproteins..tre[u Srr..clFurc,Cenet 3 '!Ol-311.

A. Jonardhan and S, UajdaMoult J, IalncsMNC. 1986.An algorilhm for dc!.rmining ihc coiforftation ofof polypcFide sesnEnts n proFiDs by sylcmadc scatch. Pmains Stl'/clFsnct C.n t ltl46-163.Nauchitelv. villlvcrde MC, SussnnnF. 1995.Solvcnt occes$ibilityas a prc-dictiv. tool for the fr. cncrgy of inhibitor binding Do hc HIV-I ProtcaltPmGin Sci 411356-13*.NicholbA, Shlrp KA, HoDigB. l99l Pmcin folding and associrtion-itsigbts

ftotn lhe inGrtacial tnd oErmodynanic proP.dies of hydrocatbons.Pm-rcins Sttuct Ffid G.nct I I .281-296.Novohy I, Bruccolcri RE Saul FA. 1989.On lhc atuiboiion ofbinding cn rgvin lhc andgen-{tibody comPlcxsMCPC603, DI.3 and HyHEL-5 ,to-che''ktrr' 28'4155-O49.Novotny ,, RlshrnAA. Eoccolcri RE. 1988.Criteria thlt dilcrimidte bctweennativeprotinsand nconlcty IoldEAnr|,dds. Pnteias Sttuct FunctCeBt4:19-30.PErk B, trvitt M. 1996. En.rgy functiors thal discrimin&tEX_rayand nc{-nativefotds from $ll-con$.ucrct d.6fs J Mol Biol 25E,361-392Pel.qucr ,L. Chens\Y. l9?. Doer conformationalrc. cnergydislitguish loopc;oformationsin protcins? Biophr! J 73t2159-23'15.Pictcn SD, SrcrnbergMIE. 1993.Etnpirical scaleof sid-chaio onformstionalcntropy in pio&in foldinS. J MoI AioI 23 :825-839Schifr.r CA. caldwcll Jw. Kollfian PA, Sttoud RM 1993. Protinsul,ctureDrcdicdonwfth a combitd solvntion recaErgy-molccularr|cchsnics otc'-fiel/d.,Molec Sit ul lotl2l-134.Sh.{ke A, Ruplcy JA. l9?3. Envircnmcnt and expoaurc o solvetrtof poGinrtoms. Lyso.ytnc and nsulir'. Biochenistty 791353'371Sippl MI. 193. Bolumaln's Principle,howledgc-ba.srlme{n fields' andpru-,il tolding. J Conputet'Aid.d MoI2culu Desisd 71413-5O1.Sippl rlj. 1995.Knowledgc-brsd otefilials for pro&i^s. Cun Opln Stru.t Bit'lSmith KC. Honis B, 1994.Evaluationof th conformational rce enrgid otlooDson DroLinr. P.otri,t' Sttu.l FaactCin t 18tll9-112SrinivaianR, Rosc CD. 195. Linus: A hicrdrchicFocedu.c to predict ie foldof a uotcin. Ptot ittr 5lr|r.r Funct Cedet 22t8t-99vrjda S. Jafri MS, Scznnln OU, Dclisi C. 193. Neccsssrvcondilions fot_avoiding incontcl polypcptide folds in confom&donal searchby t.rgyminimirrtion.aJolrbmtr.. J3:173-)92.vajda S, Sippl li4 Nbvooy J. 1997. Elnpiricsl po&ttials and tundions fo'pmt in f;Uing andbinding Cutt Opin Stnlct Biol 71222-228.vajda S, Wenc Z, Delisi c. t995. Exaacdtg hvdrophobicitygaramcteBrothsolutepariition and protcin motation/unfolding .xpetiments Prt?i'n Erli8: oEl-1092.vaidaS. wenq e Roslnf.ld R, Del-isi C. 1994 Effect of conformutiod ncx-- ibilirv anJsofvnion on reccptor_ligandbinding frt n('|6tc5.Bi&h.'r'JstryJJ:1397?-13988.v.r*hivkcr G, App.lt K, Frccr ST,viUafrancaJE. | 995. EmPiricd fr.t encrSlcalcularions f fi8aod-Prolrin crystallogilphic cot[plcis. l. Knowldge-based i8lodirD;in int mclion potdtials applicd!o E g(Edictionof-Hri_rnan rnmunodchciency rus I plst ale birding aftinity ProtainEnF8i611-69t .Wallavist A- Jcmiran RL. Covcll DC 1995. A pEferencc_barcdree cortyprramcrcrizarion f enzymc'inhibilorbinding APplicatio6 to HW-l-protcascohibitordcsign.PtDt?a, di.t:1881-1903.WcngZ, Delhi C. Vajda S. 1997.Empiiical frce nergvcalcuhtion:ComPat-ison ro calorinreiricdaltr Prurtn S.i 6:19?6_19E4WengZ Vajda S,Delhi C. I 996 PlEdictiooof complex.susingempirical rcecnrgy unctions.Pmt ilr S.i 5:614-626WcssonL EisanbeiSD. 1992.Aomic solvationpatanleErsa9pli'd to molcc-olar dynamicsoi pmtins n solutioo. Pr"rrin Sei I :227-235.Wilson C. Dotfach SA. 1989 A compullr model to dynamicallysimulatePro-tein foldin!: srudieswit cr.mbin, P,{,tein$StructFtnd Aenet 61191-209'Yuc K. Dill LA. 1996.Folding proEins wilh ! simpl! cncrgy function 6M.xEnsive oonformstioml sesrching.PruteinSci 5;254-161.

selecting near native conformations in homology modelling

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