Protein DNA Complexes: Specific

Transcription

1 Protein D oplexes: Specific rk Struch, University of ryln, Bltiore, ryln, US Sequence-specific D-bining proteins recognize n interct with iscrete bse sequences in the genoe to regulte funentl etbolic processes. Introuction Sequence-specific protein D bining is process funentl to life on erth. Fro the ientifiction of repliction sites on the chroosoe to the ifferentil expression of genes uring evelopent, ultitue of sequence-specific D-bining proteins regulte the funentl biocheicl processes of life. Unerstning how these ifferent proteins re ble to fin n bin selectively to only one,or just few,specific sequences out of the illions present in genoe is jor gol of oleculr biology. D Structure Fro the Stnpoint of Sequence Recognition Protein bining to specific D sequence entils ore thn ere recognition n interction with the liner rrngeent of the constituent bse pirs (irect reout). The precise three-iensionl structure of the region of the D olecule contining the bse pir-specific eleents tht cn potentilly interct with the protein ust be tken into ccount. This so-clle structurl presenttion of potentil recognition surfces on D cn ply criticl role in protein D bining interctions (Ki et l., 1997; Kouelk,1998). The shpe of D helix is never soothly unifor structure ue to the effect of priry sequence on such preters s tilt,roll n twist ngles of bse pir steps n propeller twisting between bses of pir. Sequence-epenent vribility of these preters long the D olecule les to loclize vritions in the with n epth of the jor n inor grooves n the propensity of soe regions to ssue noncnonicl B for,to be ore esily istorte or even to opt n intrinsic ben or kink. Becuse ifferent bse sequences ictte ifferent sptil positionings not only of interctive sites on the bses theselves (e.g. hyrogen bon onors n cceptors),but lso of tos in the sugr phosphte bckbone,theoreticlly it is possible tht specific sequence recognition coul be chieve without ny contct between protein n the bse oieties (inirect reout). owever,there hs yet to be foun cler-cut exple of sequence-specific D-bining protein tht chieves. Introuction Seconry rticle rticle ontents. D Structure Fro the Stnpoint of Sequence Recognition. Points of Recognition in the jor n inor Grooves. verview of D-bining otifs for Sequence-specific Bining. Exples of ino ci ucleotie Interction. Biologicl Rnges of Dissocition onstnts for Sequence-specific Protein Bining to D. echniss for Sequence Loction; Effects of onspecific Sequences. Role of ultiple Subunits in D-bining Proteins. oncluing Rerks high-ffinity recognition bse solely upon n inirect reout echnis. evertheless,unerstning sequence-specific recognition by ny D-bining protein ust tke into ccount the contributions of both irect n inirect reout echniss (rrington,1992). Fctors beyon priry bse sequence cn lso influence the three-iensionl chrcteristics of D regions n thus ffect the structurl presenttion of specific recognition sequence. hnges in the egrees of loclize supercoiling,chrotin copction,osotic forces n ionic strength re ong the fctors tht cn lter the presenttion or vilbility of sequence in the cell. itionlly,other boun proteins y ffect D regions sptilly reove fro their own bining sites n so ffect the presenttion of other recognition sequences. f course,cheicl oifictions (such s covlent ethyltions t certin bse positions) of oieties in recognition sequences chnge D structure by efinition n obviously cn hve profoun effects on bining interctions. Structurl presenttion y be thought of s hving n influence pririly on initil selection n iscriintion of sequences. owever,ost D-bining proteins introuce significnt chnges in D structure (reltive to the unboun stte),soe of which cn be quite spectculr (Ki et l.,1993). It is becoing cler tht the bility of given D sequence to be structurlly efore into n lternte confortion plys crucil role in ny sequence-specific bining interctions (Lesser et l.,1993),presubly ue to fvourble energetic effects cuse by incresing the interfcil copleentrity between D n protein,the ltter usully unergoing confortionl chnges s well (ege et l.,1998). The bility of specific D sequence to be efore ppers to be n especilly iportnt recognition echnis utilize by soe D-bining proteins showing strict EYLPEDI F LIFE SIEES / & 2001 ture Publishing Group / 1

2 Protein D oplexes: Specific sequence selectivity,such s restriction enonucleses n ethyltrnsferses. Points of Recognition in the jor n inor Grooves Sequence specificity vi irect reout echnis entils noncovlent interctions (hyrogen boning,electrosttic forces,slt briges,vn er Wls contcts) between the functionl groups of the ino cis or bckbone tos of the protein n the functionl groups vilble on the bses in the jor n inor grooves of the D. ssuing tht the norl Wtson rick hyrogen boning hs not been isrupte,ech of the four types of bse pir (T,T,G,G) presents istinct pttern of hyrogen onor,hyrogen cceptor n hyrophobic groups vilble for boning (Figure 1). protein,by using two hyrogen bons n intercting in the jor groove, coul esily iscriinte between the four possibilities. In the inor groove,the presence of hyrogen onor fro the 2 ino group of gunine cn be utilize for ifferentition of G or G fro T or T. owever, ny iscriintion of G fro G or T fro T in the inor groove woul hve to involve highly sensitive ens of istinguishing either the slightly ifferent steric positioning n spcing between tos or ifferentition between the slight ifference in hyrogen bon energies to the 2 of pyriiines versus the 3 of purines. Interestingly,there is evience tht such selectivity is possible (Wong n Bten,1994; Kielkopf et l.,1998). By efinition,sequence-specific D-bining proteins show egree of selectivity or preference for bining certin bse sequences over others. In ny (but not ll) cses,exintion of ifferent sites boun by protein revels reily ientifible consensus sequence responsible for recognition. For proteins recognizing sequences vi irect reout,these consensus eleents ust reflect preferre sptil rrngeent of ifferent functionl groups of the bses. In the cse of inirect reout,the consensus sequence ust be specifying prticulr n unique three-iensionl D structure not rnoly foun. owever,it shoul be kept in in tht ost,if not ll,sequence-specific D-bining proteins utilize cobintion of irect n inirect reout echniss. Soe proteins,such s certin restriction/oifiction enzyes,exhibit very strict consensus recognition eleent n re unble to tolerte even single istch. G G e e 3 3 T T Figure 1 Points of recognition in the jor () n inor () grooves of D for ech of the four bse pirs., electron cceptor;, electron onor; e, ethyl group. yrogen boning in bse pirs is inicte by she lines. in circles enotes the eoxyribose-phosphte bckbone of D. 2 EYLPEDI F LIFE SIEES / & 2001 ture Publishing Group /

3 Protein D oplexes: Specific (Two cvets shoul be entione with respect to these D-bining enzyes. First,bining ffinity shoul not be equte with enzytic ctivity: EcoRV ppers to bin eqully well to ost sequences but only ctlyses clevge t GTT sequences ue to unique cheicl properties n structurl efortions tht this stretch of D cn unergo uring the trnsition stte of ctlysis. Secon, chnging environentl conitions,such s ltertion of slt concentrtion or p,cn relx specificity.) t the other en of the sequence-specific bining spectru re proteins whose erive consensus recognition sequences show egenercies t nuber of positions n cn bin with high ffinity to bro,but not rno,subset of bse sequences. Degenercy in the consensus coul iply nuber of ifferent chrcteristics bout the interction of the protein with bse pir-specific recognition points t tht position. (1) n ino ci sie-chin ight interct with ifferent bse pirs with little or no steric occlusion presente by other functionl groups in the vicinity; for exple, hyrogen onting group boning with the 7 position of purine regrless of whether 6-ino group () or 6-keto group (G) were present. (2) The protein ight hve the flexibility to ssue ifferent confortions (or inuce ifferent D confortions) in orer to just the sptil positions of intercting groups; for exple, hyrogen-ccepting sie-chin intercting with the 4- ino group of cytosine whether fro G or G pir t prticulr position. (3) Liite egenercies ight inicte positions where the bses o not provie functionl groups irectly intercting with the protein but where certin bses ffect the structurl presenttion of recognition points in neighbouring positions. onversely, the invrint occurrence of specific bse t given position oes not necessrily inicte tht it contributes groups tht interct irectly with the protein: it coul be strictly require for structurl presenttion of other intercting eleents. (4) Degenercies ight lso be seen t positions where certin bse cn contribute irect points of contct,but the bsence of such contcts oes not ffect the overll bility of the protein to bin,given tht enough other contcts re vilble t other positions. owever,such situtions woul probbly result in the bining interction hving ifferent theroynic or kinetic properties (i.e. ltertions in DG,issocition constnt,on or off rtes,etc.) epening upon the ctul D sequence. lthough it y be possible to efine one prticulr sequence vrition s hving optil recognition points n bining stbility vi in vitro biocheicl or biophysicl esureents,this shoul not be confuse with the concept of optil in vivo functioning,which is ultitely the result of protein n D-bining site coevolving to ccoplish physiologicl role within the constrints of the cellulr ilieu. verview of D-bining otifs for Sequence-specific Bining ny sequence-specific D-bining proteins hve been groupe into filies bse upon the type of structurl otifs use for recognition n interction with their D trgets. vriety of clssifiction schees hve been evise,ech involving soewht ifferent groupings,but n initil bro clssifiction cn be e on the bsis of which type of protein seconry structure is use for recognition: helix, b sheet or soe type of loop. Further subivision into specific filies is usully bse upon the nner in which the recognition eleent is sptilly relte to the surrouning protein structure or the etho of ultieriztion between iniviul subunits in ultieric protein. ot ll known D-bining proteins cn be unbiguously plce into fily grouping n there is no reson to ssue tht ll filies hve been iscovere. t present,generlly gree upon jor filies of otifs inclue the helix turn helix (T), hoeooin,f-3/fkh winge helix,zinc fingers,zinccontining steroi receptors,leucine zipper,helix loop helix (L),Rel n b ribbon. Since etile escriptions n iscussion of these jor filies cn be foun elsewhere in these volues n in sources cite t the en of this entry,only few generl principls will be briefly touche upon here. The jority of sequence-specific D-bining proteins exine,incluing ebers of the T,hoeooin,steroi receptor,winge helix,n zinc finger filies,eploy helices for recognition. (Leucine zipper n L proteins contin bsic oin rich in rginine n lysine resiues tht ppers to be only prtilly helicl in solution but which unergoes confortionl trnsition into typicl helix upon bining to D.) Usully, the so-clle recognition helix intercts with specificity eterinnts in the jor groove. owever, vriety of wys for inserting recognition helix into the jor groove hve been observe,even ong ebers of the se fily. For exple,the bcteriophge li n Escherichi coli TrpR proteins re both T fily ebers,yet the recognition helix of I lies lengthwise in the groove while TrpR orients its recognition helix perpeniculrly in the groove. It shoul be ephsize tht no single recognition helix cn in n of itself bin to D in sequence-specific nner. three-iensionl protein rchitecture is require not only for proper positioning of the recognition helix,but lso to provie itionl D contcts,either to the bses or the phosphte bckbone, which re necessry for bining stbility n ccurte sequence iscriintion. In fct,the notion of protein hving its sole bining specificity eterinnts present within single oin or otif is rther isleing: ultiple otifs or contct oins re usully require for high-fielity sequence specificity. The occurrence of ore EYLPEDI F LIFE SIEES / & 2001 ture Publishing Group / 3

4 Protein D oplexes: Specific thn one otif,either of the se or ifferent type,is often the result of ultier fortion,but ultiple otifs y lso be present within single polypeptie chin. b Sheets rrnge in ntiprllel fshion (b ribbons) hve lso been observe to function s sequence-specific recognition eleents vi interctions in the jor groove. itionlly,there re exples where b ribbons ke sequence-specific contcts within the inor groove. The coponent b sheets foring the ribbon cn either be present on the se polypeptie (s in the cse of the eukryotic TT-bining proteins) or coe together vi ieriztion of ienticl polypeptie chins (see below). Recently, new otif which relies upon neither n helix nor b sheet for sequence recognition hs been efine by structurl stuies of vriety of eukryotic trnscription fctors involve in cellulr stress responses n evelopentl events. ebers of this fily,clle Rel,utilize projecting hyrophilic loop of polypeptie to ke sequence-specific contcts with five contiguous bse pirs in jor groove (hytil n Verine,1996). ny D-bining proteins use vrious other types of loops n strns to ke D contcts,in ition to the contcts e by the jor recognition eleents. The contribution to overll bining energy provie by these itionl contcts is usully bsolutely necessry for the stbility of bining. For the ost prt,the heterogeneity in structure of these loops n strns prevents the fro being esily groupe into efine clsses of otifs. nuber of vritions on few thees hve evolve to chieve sequence-specific D recognition by proteins. lthough ny otifs cn be groupe into filies,ech D-bining protein is unique. It will be especilly interesting if future reserch les to the iscovery tht ifferent proteins,either nturlly occurring or constructe using protein engineering techniques,cn recognize the se D sequence with coprble ffinities using entirely ifferent types of recognition otifs (e.g. helix versus b ribbon). Exples of ino ci ucleotie Interction oncovlent bons e by ino ci sie-chins or the in chin of the protein (usully the ies) to the bses n phosphte bckbone of the D trget unerlie ll sequence-specific interctions. The jority of contcts re usully e vi hyrogen bons but vn er Wls contcts n electrosttic interctions cn occur n re often criticlly involve. f prticulr interest for ost sequence-specific interctions re the contcts e between the ino ci sie-chins n the bses. Given proper context,it ppers tht nerly every type of free sie-chin cn ke soe type of contct with t lest one of the four bses. Prcticlly every polr ino ci hs been observe to be cpble of king bse pir contct n soe exples re shown in Figure 2. itionlly, nonpolr hyrophobic sie-chins of lnine n isoleucine hve been seen to ke vn er Wls contct with the 5-ethyl group of thyiine,n even the rotic ring of phenyllnine is interclte between bse pirs in soe coplexes. o generl recognition coe of one-to-one corresponence relting specific ino ci to single specific bse with which it cn interct exists. ny ino ci siechins cn interct with ore thn one type of bse n ny given type of bse cn be contcte by ifferent sie-chins. ften,ore thn one sie-chin contcts given bse,n in other instnces single sie-chin cn contct ore thn one bse pir siultneously. lthough no siple rules of recognition hving pplicbility to ll sequence-specific interctions see to exist,there y exist coes governing the interctions seen for ebers of soe filies,in prticulr the zinc-finger proteins (hoo n Klug,1997). yrogen-boning interctions between protein n D nee not be irect: frequently they entil one or ore wter-eite contcts. The extensive n iportnt role tht wter plys in sequence-specific protein D bining hs been one of the ost surprising n exciting results of recent structurl eterintions. For exple,in the crystl structure of the bcteril Trp repressor protein boun to its cognte D trget,the iportnt contcts between resiues of the recognition helix n bse pirs require for sequence specificity were ll observe to be eite vi orere wter olecules, fining confire by ny subsequent stuies. fluctuting vribility in positions n boning interctions of wter olecules eiting contct between the ntennpei hoeooin n its D trget rticlly illustrtes the ynic nture of protein D interctions,even in stble coplexes (see Schwbe,1997). Biologicl Rnges of Dissocition onstnts for Sequence-specific Protein Bining to D vriety of in vitro ethoologies re vilble for esuring the theroynic n kinetic properties of protein D bining. The ost coon,n frequently esiest,preter to erive is the pprent equilibriu issocition constnt (K ) of the overll interction. K esureents e in vitro re extreely useful for ressing nuerous questions when exining prticulr sequence-specific bining rection. owever,cution ust be exercise in interpreting these results s being truly reflective of in vivo theroynic properties since the experientl conitions use to erive K vlues ber little reseblnce to intrcellulr situtions (in vitro,sll 4 EYLPEDI F LIFE SIEES / & 2001 ture Publishing Group /

5 Protein D oplexes: Specific () (b) 2 3 G T (c) 2 () 3 T 2 S G Figure 2 Exples of ino ci bse pir interctions. () rginine boning to gunine. (b) Serine boning to enine. (c) sprgine boning to enine. () ysteine ouble contct to enine n gunine of jcent bse pirs. In (), the enine is ttche to the opposite strn reltive to the gunine but the bses hve been rwn on the se plne. yrogen bons re enote by she lines. efine pieces of D contining the trget sequence re use n thus the trget is not within its nturl surrounings subject to chrotin conenstion n pckging forces n vritions; rrely re other Dbining proteins or enzyes present,let lone the ultitue present in nucleus or cell; ionic n osolrity conitions re usully substntilly ifferent fro those in vitro,n so on). Unfortuntely,ccurte theroynic ssessents of specific protein D bining rections in vivo re not possible using current technologies. The bove cvets being given,ost sequence-specific protein D coplexes hve K vlues in the rnge of ol L 2 1. In generl,proteins with ore strict sequence specificity,such s bcteril repressors cting t one or just few sites in the genoe,exhibit higher ffinities in vitro (i.e. hve lower K ) thn o proteins with ore relxe sequence specificity. In cses where resonbly ccurte ssessents re possible,the ifference in K vlues for specific trgets versus rno D (the sequence iscriintion fctor) is in the rnge of echniss for Sequence Loction; Effects of onspecific Sequences Before stbly bining t specific D sequence,the protein ust first locte the sequence rpily n selectively fro ong the illions of possible sequence peruttions present in the genoe. It is unresonble to believe tht this coul be effectively ccoplishe by rno collisions of the rectnts in proper orienttion. s llue to bove,ll sequence-specific D-bining proteins possess egree of nonspecific ffinity for D, pririly through electrosttic interctions with the sugr phosphte bckbone. The free energy coponent llowing these nonspecific bining interctions is believe to be ostly entropic in nture n erive fro counterion isplceent fro the bckbone s well s the isorering of wter olecules tht norlly hyrte the D. onspecific ffinity llows the protein to rpily sple sequence peruttions,presubly by soe type EYLPEDI F LIFE SIEES / & 2001 ture Publishing Group / 5

6 Protein D oplexes: Specific of sliing echnis long the D n juping between proxil segents. When the protein encounters its specific trget n intercts with sequence-specific recognition eterinnts,the electrosttic interctions with the sugr phosphte bckbone responsible for the nonspecific bining oe still ply criticl role: they contribute significnt ount of the fvourble bining free energy without which stble coplex fortion woul be ipossible. In ny lrge genoe,the chnces re tht nuber of sequences resebling specific bining protein s recognition site exist n tht they hve soe egree of intereiry ffinity for the protein. nuber of scenrios cn be envisione whereby evolution coul hve exploite these seispecific sites for regultory purposes. ongregtion of seispecific sites ner prticulr physiologicl trget site coul hve evolve s ens to increse the loclize concentrtion of the protein ner tht trget n so ensure ifferentil probbility of protein bining there versus t trget not surroune by seispecific sites. Even ore intriguing woul be if one or ore optil bining sites for prticulr protein existe but were inccessible uner soe conitions (ue to soe fctor such s chrotin conenstion) n the protein ws exerting regultory effect by bining t suboptil sites uner those conitions. If the optil sites bece ccessible in response to soe cell cycle or environentl signl,then they ight serve s sink to titrte the protein wy fro the suboptil sites n thus chnge the regultory effect. Role of ultiple Subunits in Dbining Proteins The jority of sequence-specific D-bining proteins exine to te re ultieric in solution (often either hooieric or hootetreric) n in these cses ultieriztion is usully necessry for high-ffinity bining. owever,there re ny exples of sequencespecific proteins tht bin s onoers so ultieriztion is not n bsolute requireent for high ffinity. (Soe,but not ll,of these onoeric biners o possess two or ore seprble recognition otifs in the se polypeptie.) Reltive to onoeric single site interction,using ultiers to recognize ultiple jcent sites woul obviously increse the overll nuber of protein D contcts in the coplex n so woul be expecte to increse bining strength. It is not ifficult to igine tht ny hooultieric D-bining proteins were once onoeric single site biners tht coevolve with their trget sites uner selective pressures fvouring tighter bining. There is lso source of recognition flexibility n vribility ttennt upon the use of ultieric Dbining proteins. For exple,ebers of the leucine zipper n helix loop helix filies of D-bining trnscription fctors cn for heteroiers with other ebers of their own fily. Since ech iniviul onoer brings with it ifferent D-bining properties, heteroier fortion s egree of regultory verstility tht cn be ltere by chnging the reltive expression levels of the subunits in response to ifferent stiuli. For soe proteins,prior ultieriztion is n bsolute requireent for sequence-specific D bining: the iniviul onoers hve no D-bining bility n ultieriztion is necessry to ctully for the D recognition/bining otif. The b-ribbon fily ebers etj,rc n nt re excellent exples of this ctegory. ultieriztion is the bsis for coopertive bining behviour exhibite by ny sequence-specific Dbining proteins. Bining coopertivity cn result fro ultier fortion t two ifferent stges: ssebly of the ultier prior to D interction or ssebly of the ultieric coplex on the D. For the forer,rtic coopertive effects woul be observe if the issocition constnt for protein ultieriztion ws significntly greter thn the issocition constnt for the ultier D bining rection. In the ltter cse,coopertivity coul be ue to either confortionl chnge in the protein entity initilly bining D which kes it better trget for further protein protein interctions necessry for stble coplex fortion,or ue to confortionl chnge in D structure jcent to the initilly boun protein which cuses tht prticulr stretch of D to be better substrte for itionl protein bining (Vshee et l.,1998). In these ltter scenrios,the initilly boun protein entity coul be either onoeric or lrey ultieric,n either hoologous or heterologous proteins coul be the subsequent biners. oncluing Rerks Sequence-specific D-bining proteins interct with sptil rrngeents of tos n rective groups on D which re specifie either by prticulr sequence of bse pirs or by closely relte peruttions of sequence. In the pst few yers structurl stuies using X-ry crystllogrphic n nucler gnetic resonnce (R) techniques hve elucite the structure of ny Dbining proteins in both the free n D-boun sttes. welth of infortion concerning how proteins n D interct to for stble,high-ffinity boun coplexes hs been obtine. Further structurl stuies on ifferent clsses of D-bining proteins n utnt vrints will provie new n eeper insights. It ust be borne in in, however,tht protein D interctions re ynic processes tht cnnot be fully unerstoo erely by solving sttic or equilibriu structure. jor irection 6 EYLPEDI F LIFE SIEES / & 2001 ture Publishing Group /

7 Protein D oplexes: Specific of future reserch will be not only to issect the biophysicl n theroynic preters responsible for stble coplex fortion,but lso to integrte this infortion with knowlege bout how chnges ffecting these preters cn bring bout physiologicl chnges in gene expression n D etbolis controlle by ifferent sequence-specific D-bining proteins. References hoo Y n Klug (1997) Physicl bsis of protein D recognition coe. urrent pinion in Structurl Biology 7: hytil n Verine GL (1996) The Rel fily of eucryotic trnscription fctors. urrent pinion in Structurl Biology 6: rrington RE (1992) D curving n bening in protein D recognition. oleculr icrobiology 6: ege RS,Wng -F,Ki SS n Schpir (1998) Subunit rerrngeent ccopnies sequence-specific D bining by the bovine ppillovirus-1 E2 protein. Journl of oleculr Biology 276: Kielkopf L,White S,Szewczyk JW et l. (1998) structurl bsis for recognition of T ntbse pirs in the inor groove of B-D. Science 282: Ki E,lbrechtsen n Deppert W (1997) D-confortion is n iportnt eterinnt of sequence-specific D bining by tuor suppressor p53. ncogene 15: Ki Y,Geiger J,hn S n Sigler PB (1993) rystl structure of yest TBP/TT-box coplex. ture 365: Kouelk GB (1998) Recognition of D structure by 434 repressor. ucleic cis Reserch 26: Lesser DR,Kurpiewski R,Wters T,onnolly B n Jen-Jcobson L (1993) Fcilitte istortion of the D site enhnces EcoRI enonuclese-d recognition. Proceeings of the tionl cey of Sciences of the US 90: Schwbe JWR (1997) The role of wter in protein D interctions. urrent pinion in Structurl Biology 7: Vshee S,elcher K,Deng WV,Johnston S n Koek T (1998) Evience for two oes of coopertive D bining in vivo tht o not involve protein protein interctions. urrent Biology 8: Wong J n Bten E (1994) TBP D interctions in the inor groove iscriinte between :T n T: bse pirs. ucleic cis Reserch 22: Further Reing Berg G n von ippel P (1988) Selection of D bining sites by regultory proteins. Trens in Biocheicl Sciences 13: Gehring WJ,ffolter n Burglin T (1994) oeooin proteins. nnul Review of Biocheistry 63: rrison S n ggrwl K (1990) D-recognition by proteins with the helix turn helix otif. nnul Review of Biocheistry 59: Lilley D (e.) (1995) D Protein Structurl Interctions. xfor: IRL Press. Pbo n Suer RT (1992) Trnscription fctors: structurl filies n principls of D recognition. nnul Review of Biocheistry 61: Runn BE,Brown B n Suer RT (1994) jor groove D recognition by b-sheets: the ribbon helix helix fily of gene regultory proteins. urrent Biology 4: Trvers (1993) D Protein Interctions. Lonon: hpn n ll. EYLPEDI F LIFE SIEES / & 2001 ture Publishing Group / 7