SCIENTIFIC
`
`[E N ZY M Ii M OLICCU LE.
`
`152'] 7’) ' 6'15} 175'
`
`I966)
`
`Apotex Inc. et a]. V. Amgen Inc. et a ., R2016—01542
`Page 1
`
`Am n Exhibit 2011
`
`

`

`The Three-dimensional Structure
`
`of an Enzyme Molecule
`
`‘The tzrrarzgermerit of arrmis in an £’!l:__t-"tn? rnolecule has been. wot’/md our
`
`for the _fir'.s't time. The c*r::_-wnt» is l_y‘.s0.-‘:._jv*:ne, whiz-I2 firm/£.s' open r_.'t’H.s'
`
`of l)(2(‘f€I‘t(t. The .s't1:d__v has also shoum. how l_j,'so.:_-1--'rrze perfornzs its task
`
`no (lay in 1922 :\lcx;mtlcr Flem-
`
`Oing was sailleriirg from n cold.
`
`This is not unusual
`
`in London.
`
`by lluvial
`
`(2. Phillips
`
`rletermiued and whose properties are
`Itmlnrstoml
`in atomic detail. Among
`these properties is the way in \\.’l.'l.l(’.'l'l lhe
`enzyme combines with the substance on
`which it
`ac-l:«'—:1 complex sugar in the
`wall ol the lmcterial cell.
`
`Like all cnz_vme5. lysozynic is it pro-
`tein.
`Its Chcniical mnlteup has hccn
`established
`Pierre jollés and his
`rcollezigues at
`the Unix-‘ersity of Paris
`and by Robert E. Cmifielsl of the Co-
`lnmhin Uniw.-rsil_v College of Pliysiciaiis
`nntl Stlrgeoras. The_\-' have lound tlmt
`em.-h molecule ol
`lysozyuiic ohtninccl
`from egg white consists of a single
`[J0l_vp€pliIle
`cllnin of 129 amlinu tt(.'l(l
`snhculit.-a of 20 djllurrmil
`lcinds. A pep-
`tide homl
`is lonnetl wlien two amino
`
`acids are joined following the removal of
`.1 molecule of waiter. It is cnstornury to
`Citll
`the portion of the amino ncid in-
`
`corporsitcd into It polypeptide chain .1
`residue, and each residue has its own
`c-lnm-wteristir: side t.-lmin. The l29-re.-:i-
`
`due l_\'.'iozyIne molecule is cross-linlced
`in
`four places by tlisullida bridges
`ltJl"l11tE'.(l by the cornhinntion of sulfur-
`:-ontuiuing side chains in tlifferent parts
`of the rnolecnlc [arm illrrslrafiort on up-
`pnsilte prrgc].
`The properties of the molecule cauniot
`he uuderstu-otl
`from its chelnicnl L-on~
`
`sfitntion alone; they depend most criti-
`cally on what parts of the niolecule are
`brought close together
`in the loltletl
`tl11'ee-dimensional stnir.-t'ure. Some form
`
`of microscope is rleederl to t'3ittl.Illl‘lt! the
`structure ol
`the molecule. Fortunate-
`
`ly one is effet-tively provitlecl by the
`techniques of X-my crystal-.-rtmcturo
`nlnllysis pioneered by Sir Lawrence
`Bragg and his father Sir William Bragg.
`
`ALA ALAN NE
`ARG nRGiNIr~.IE
`ASN ASPAR-’\_(;~INF
`ASP a1SP.€‘..!R1'C
`AC.J.‘.=
`CYS CYSTEINE
`GLU ‘3LL_lTJ\M|C I‘-CI|‘J
`GLN [—JL1,llI'-\l'U'l|l\.'E
`
`GLY r.aLrIfI|‘uE
`HI5 H-STI:’J:NF
`ILEU :5I.Z:LE'_IJr;'sN+_
`LEU |l-Il|l'|u"Jl.-‘
`LYS l.r§5.il\It
`MET I".»tErHsf_It».'-INK
`PHE i4'HEN'r1..mAN=r‘.IE
`
`PFID I“RO1.IN':
`SER SFRINE
`THR 'IHl-.’l:+'_lNil\.'|-_
`TRY lH‘fl-"|{JPHAN
`TYR ivHt)S:Nl-_
`VAL W-[INF
`
`TWO-DINIENSIDNAL MODEL 0! the lywzyme molecule lit uhown on the opposite page.
`Lyauzyme in a protein containing 129 amino in-ld mhunitr-. rumnlnnly culled residues. Iaee
`key to abbreoiutiams nbm.-e I. These rcriducs form n polypeptide chain that in rrose‘-linked in
`four |Ilm:'l:4! by dirulficlu I —t-‘-—5-
`I hands. The amino arid acqnentre of lywzp-me was. deter-
`mined iudepulidently Ivy Pierre lulléu and his co-workers at the University of Paris and by
`Robert E. Cnnlield nl the (Zulumhia University College of Physiciuniu and Surgeons. The
`tl-n-on-rlimnnninmul structure of the ly:-ozyme molecule has now been established with the
`help of X-rny crjmtullugraphy by the null-nor and his colleague: at the Royal Institution in
`London. A painting of the molt.-rule's three-dimensional structure appears on pages 80 and
`8]. The innrtinn of lyaozyma is to unlit n pnrticulur long-chuin mull.-rtlle, n rorupla.-Jr. sugar,
`found in the outer membrane at many living cells. Molecules that are acted on by enzymes
`are known as substrates. The sulnatrale of lysozyme lit!» into : cleft. or pocket. Iorlued II)’ the
`three-dintenuionul structure of the lysosyme molecule. In the two-dimensional model on
`tho opposite [Iago the amino acid rnidueu Illll line the pucluet. are ulwwn ill Llurk green.
`
`but Fleming was :1 most unusual man
`and he look zulvauitngu of the cold in at
`nhnrnctcristiL- way. He allowed in few
`drops of his nasal mucus in fall on a
`culture of bacteria he was working with
`and then put the plate to one side to
`see what would happen.
`lmnginc his
`excitement when he di.-zcoverecl some
`time later that
`the haeterin near tlm
`
`mums land dissolved nway. For in while-
`hc thought his nnihition ol Hurling n
`IltIi\’t:t‘:tal.l aintihiotic had he-(en rcnli:r.cd.
`
`(_'.‘iltllJ-
`lllIlCl(l_\-‘
`lll ll burst of activity lIl.’.
`lixhed that
`the :'tI|lll)‘¢lClt':tt‘lill nction of
`
`the inn:-u.-: was tlnc to the l'1I“F.'St?II(,“l" in
`it of an enzylnc; he ullletl this sI.Il)sta1Iwc
`lysozymc because of its c:1pucit_v to lyse,
`or dissolve,
`the l)n(:t-Brinl cells. Lyso-
`zyrm: was soon (ll.’1't:(l\’<:l’t:tl
`in nmny tis-
`slms and secrelimis of the lnnnan lt-tJll_\-‘.
`in plants and most [alentifully of nll
`in
`the white of egg. Unfortunately Flem-
`ing found that it is not cllcctivc against
`the most harmful bacteria. He had to
`
`wait seven years before u strznigoly
`similar experiment
`revealed the exis-
`tence of :1 genuinely effective .-1ntihi-
`otic: penicillin.
`Nevertheless, Fleming's lysozynm has
`proved It more vnluulnlc discovery than
`he t.-on liavc I_'1\‘p£'.t'.'l.£'t.l wlicn its prop-
`crticsi were first cslnhlislietl. With it.
`
`for example, bacterial nnntornists have
`been uhle to study many tletatils of ham-
`terial structure [see "Fleming's Lyso-
`zyme," by Robert F. Acluar and
`E.
`1-Inrtsell; SC'.IE.\"I'lI-'1C AMERICAN,
`june,
`1960].
`It has now tumed out
`that
`Lvsozyrne is
`the first enzyme wlmse
`three-tlimcnsional
`stnacture has been
`
`3'3
`
`
`
`Page 2
`
`

`

`CAHBOJRYL END
`
`r
`
`H
`
`\N—
`/
`
`H
`AMINO END
`
`‘G
`
`Page 3
`
`

`

`
`
`
`
`
`
`Page 4Page 4
`
`

`

`MAIN cnmn: * rurrnoeen
`
`cannon
`
`— oxvem
`
`
`
`'I'HRF.l{-IIIMENSION.-U. MOHEI. of the ly-
`:=II2)‘l'rI2 molecule. paints.-d 131.‘ Irving Ceis, in
`luau.-d turn an actual mm]:-I nan:-ntllllrtl III the
`Iluynl In-liltltiult h_\' Ibo Ilttthur nml Ilin nul-
`lengues. The painting enables» one In Irarc
`and tlintingninh between the 1-I11-mica] lmntis
`that hold together the main polypeptide
`chain and the |.mmle'- in the 129 side rhuim-.
`one for eaelt amino acid residue. The mole-
`cattle in iuldml .-'0 us In [arm 1.! vldaft that halt]:-
`the Hlhntrate mnlertlle uhilc it
`is Infill;
`broken in two. The painting. on the next
`page sllunms how the !-I-lllbII'IIE' fits into the
`rlnfl. The real balls represent murgelt atoms
`that urn important In splitting the substrate.
`
`SIDE cmum — SULFUR
`camaon
`
`T HYDROGEN
`BOND
`
`Page 5
`
`

`

`

`

`The dilliculiics ol examilling mole-
`cules in atomic detail arise, oi course.
`from the fact that molecule-5 are very
`small. Within a molecule each atom
`
`is usually separated from its neigl1boI'
`by about L5 angstrom units (1.5 x lit”
`centimeter). The lysmrymc molecule,
`which contains some 1.950 atoms,
`is
`almut
`-10 angstroms in its largest di-
`mension. The lirst problem is to {ind I1
`microscope in which the atoms can be
`rcsiolved from one another. or seen sep-
`amtcly.
`The resolving power of n niicroscope
`tlcpcmls lum.lanm:nlally on the wave-
`length ol
`the adiation it employs.
`In
`general no two objects can be seen: sep-
`arately if they are closer together than
`about half this wavelength. The short-
`est wavelength tran.-irnitletl by optical
`microscopes {those working in the ul-
`tmviolct end of the s“pecl'I'u::I1) is aliouli
`2,000 times longer
`t.l1an the distance
`between atoms. In order to "see" atoms
`one must use radiation with a much
`
`shorter wavelength: X rays, which have
`a wavelength closely comparable to
`interatomic distances. The employment
`cl X rays, however, creates other dif-
`ficulties: no satisfactory‘ way has yet
`been found to make lenses or mirrors
`
`that will focus then: into an image. The
`problem,
`then,
`is
`the apparently im-
`possible one of designing an X.-ray
`microscope without lenses or mirrors.
`Consideration ol the di.ll'r:u.-tion the-
`
`ory of microscope optics, as developed
`by Ernst Abbe in the latter part of the
`19th century, shows that the problem
`can be solved. Abbe taught us that the
`formation of an image in the micro-
`scope um be regarded as a two-stage
`process. First, the tlbject urldcr t.'.K.'i.l]ll-
`nation scatters the light or other radia-
`tion falling on it in all directions, form-
`ing a diliraction pattern. This pattern
`arises because the light waves scattered
`from different parts of the object com-
`bine so as to produce a wave of large
`or small amplitude in any direction
`
`according to whelller lltti waves are in
`or out of pha.-se-—in or out of step-
`with one another. {This effect
`is seen
`most easily in light waves scattered
`by a regularly repealing structure, such
`as .1
`tliflrrlction grilling made of lines
`st.-ribetl at regular intervals on a glass
`plate.)
`In the second stage of image
`formation, according to Abbe,
`the ob-
`jective lens of the microscope collects
`the dillracled waves and re-combines
`
`to form an image of the object.
`then:
`"Most important,
`the nature of the im-
`nge depends critically on how much ol
`the dillmction pattern is used in its
`formation.
`
`X-Ray Structure Analysis
`
`In essence X-ray structure analysis
`makes use of a microscope in which
`the two stages of image formation have
`been separated. Since the X. rays can-
`not be focused to form an image di-
`rectly,
`the diffraction pattern is
`re-
`corded and the image is obtained from
`it by calculation. Historically the meth-
`od xv-as not developed on the basis of
`this reasoning. but this way of regard-
`ing it {which was lir.-it suggested by
`Lawrence Bragg] brings out its essen-
`tial
`features and also introduces the
`
`In re-
`main tllflit-Illty of applying it.
`cording the intensities of the dillmcted
`waves, instead of focusing them to forrn
`an image, some crucial
`information is
`lost, namely the phase relations among
`the various dilfracted waves.
`\\'ithout
`
`this information the image cannot be
`formed. and some means of recovering
`it has to be found. This is the well-
`
`iznuwn phase problem of X-ray crys-
`tallography. It is on the solution of the
`problem that the utility of the method
`depends.
`The term "X-ray clystallogrepliy" re-
`minds us that
`in practice the method
`was developed (and is still applied} in
`the study of single crystals. Crystals
`suitable for study may contain some
`
`MODEL OF SU BSTHATE shows how it fits into the rich in the Ijrroitymc molecule. All the
`carbon atoms in the substrate are shown in purple. The portion of the substrate in intimate
`contact with the underlying enzyme is u polysucrbaricle chain corn-iating of six riugliku struc-
`turcu. each a residue o[ no amino-sugar molecule. The znubatrnle in the model is mule up of
`six identical residues of the amino sugar rolled ill-arelylglucouaminc I NAG I. In the actual
`substrate every other residue is an amino nugar known as N-ncetylronramic acid (NAM I.
`The illasuation is lined on X-my studies oi the way the enzyme is bound to l tria-at-charirle
`made. of three NAG units, which fills the top of the clelt: the arrangement oi NAG units in
`the llotlom ol the clell was worked out with the aid of three-dimensional models. The sub-
`stnte is held In the enzyme by in complex network of hydrogen bonds. in this style of model-
`meking each straight section of chain represents I bond between atoms. The alarm [hem-
`selvec lie II the intersections and elbows of the structure. Except for the [our red bull: rep-
`resenting oxygen atoms that are active in splitting the polysorcluaridc substrate, no attempt
`in m.rulc_lo represent the electron shells of atoms because they would merge into a solid mess.
`I4
`
`10'“ identical molecules in a regular
`array; in eliecl the molecules in such a
`crystal dil:lract the K radiation as though
`they were a single giant molecule. The
`crystal acts as a three-dimensiional dif-
`l'r:z(:tinn grating, so that the waves scat-
`tered by them are confined to :1 number
`of discrete dircctioris. In order to obtain
`
`a three-dimensional image of the struc-
`ture the intensity of the X rays scatterecl
`in these dil-lcrent directions must be
`
`measured, the phase problem must be
`solved somehow and the measurcnients
`
`nnnit be combined by a corn pater.
`The recent. successes of this mctllud
`
`in the study of protein structures have
`depended a great deal on the develop-
`ment of electronic computers capable
`of performing the calculations. They
`are due most of all, however,
`to the
`Lliscovery in 1953, by .\I. 1". Perntz of
`the Medical Research Council Labora-
`
`tory ol Moleclllar Biology in Cambridge,
`that
`the rnethod of "isomorphous re-
`plat.-einent" can lie used to solve the
`phase problem in the study of protein
`L‘t'_$'Sltll.-5. The method depends on the
`prepar;-ition and study of a series of pro-
`tein crystals into which additional heavy
`atoms. such as atoms of umnimn, have
`been introduced without otherwise af-
`
`fecting the crystal structure. The first
`successes of
`this method were in the
`
`study of sperni-whale myoglobin by
`John C. Kendrcw of the .\-lerlical Re-
`search Council Laboratory and in Pa-
`rutz’ own study of horse hemoglobin.
`For their work the two men received the
`
`Nobel prize for chemistry in 1969. [see
`"Tlie Three-dimensional Structure of :1
`
`john C. Kcuclrew.
`Protein .\loIcr:Iule,"
`SCIENTIFIC :‘\M1-2.H.IC..'|N, December, 1981,
`and "The Hemoglobin Molecule," by
`M. F. Perutz, Sou-':.\"I1|»'n.' ARI!-.'Itl(-JAN,
`November, 1934].
`llecause the X rays are scattered by
`the electrons within the molecules, the
`
`image ca.lcuJated from the diffraction
`pattern reveals the distribution of elec-
`trorls within the crystal. The electron
`density is usually calculated at o regu-
`lar array of points, and the image is
`made visible by drawing contour lines
`through points of equal electron den-
`sity.
`IF these contour maps are drawn
`on clear plastic sheets. one can obtain
`a three~dimensiona.l
`image by assem-
`bling the maps one above the other in a
`stack. The amount of detail that can be
`
`seen in such an image depends on the
`resolving pow:-.r of the clletrtive micro-
`scope, that is, on its "aperture," or the
`extent of the dilfmetion pattern that has
`been included in the formation of the
`
`image. If the waves dilirar.-ted through
`sufliciently high angles are included
`
`83
`
`
`
`Page 7
`
`

`

`(mrrt-.s[m:IdiIag to a Iau'ge aperh1.re}, the
`utmn.-«'
`lIl‘ll)(.’4'Il'
`:15
`individual
`[walks
`in
`the image map. At
`lower resolution
`gmups of unrcsnkecl uturns appear with
`I.'l1:IrlI(:l(fl'i5l‘iI.'.' shapes by which they can
`he l'l3('(lg!'Ii‘!.{'(1.
`The tIII‘I:!t2—(1iL1I£msim'mI
`
`Sl'I‘ll(:hIfl.'!
`
`LIE
`
`Iystrzyxnc cry:-'tal.[ized {mm the white of
`Iu-u'.~: egg has been determitled in utmw
`ic cl:-tail with the X~my metlmtl by mar
`group at the Royal Institution in Lon-
`
`j LYSOZYME. mm CHAIN
`
`dun. This is the lalmmtnry in which
`Humphry Davy and .V1ir.-luau] Felnulay
`made their Iundumentall discoveries dur-
`
`ing the 19th century. and in whiw.-h the
`X-my method uf stnwture analysis; was
`developed between the two world want
`by the brilliant gruup of wurkers led
`by William Bragg. including
`D. Ber-
`nnl, Kathleell Lnnsdale. W. T. Asthtlry.
`J. M. Rnlbertxoln am-:1 n1am_v ntlwrs. Our
`work on lysozymc \vaL-: begun in 1980
`
`when Roberto J. Puljalc. u visiting" war}:-
`er [mm Argentina. demonstrated that
`suitnhlr: t'1'ysl'uJs t,-unl.a1i1:iI1g heavy atoms
`r.-rmlcl be prepared. Since then C. C. F.
`Blake. A. C. T. Nurth. V. R. Snrma.
`Ruth Fe-ma, D. F. Koenig, Loni:-‘av N.
`juhnsun and C. A. Muir have p'w.)'e{l
`iniparlzmt rules in the work.
`In 19552 {L
`Iuw-resulutim image at
`the stmr.-tuw was nhtuine(l that revealed
`the gt-ncraal .-almpe of the Inulemulo and
`
`c:.-.-.= LYSOZYME. SIDE cum:
`
`3 suasm-us. MAIN CHAIN
`
`
`
`j suasrnxre. sun: cumu
`
`E HYDROGEN sour:
`
`T DISULFIDE BOND
`
`MM" OF LTSOZT.-\IE AND SLIBSTRATE depicts In rotor the
`Neutral I“1’IllirI of each molecule. Side rluuins have been nlnilled mt-
`mepl
`lnr llsnrw that prmltlrn Iln-. four tlisulfidr hcmrls clipping the
`Iysoxyme molerule. together and thus: that supply the terminal con-
`
`nwtiunec for lurtlmgen [loads holding the nulnltrnlc to the lynozyme.
`The tap lllree ring» of the snlnatrula IA. 3. C I are held to the un-
`derlying eluynm by six prim-ipnl hydrogen lnnnfln. which are Iden-
`tified by number In key with the desrription in the text. The lym-
`
`31-
`
`
`
`.*-‘-1.'fE;'I'I
`
`
`
`—-?-r‘-'~ :
`
`'—r.'"-\._.-——r‘.-
`
`_"a=-at-q.‘
`
`

`

`the
`til
`the arrniigerm.-lit
`that
`shuwetl
`pulyfrepficle Chain is even more enm-
`plex than it is in mynglnhin. This lnw-
`resolntinn image was ('itll.‘lIl:ltt‘tl
`lrnm
`the uniplitmles til abnut -‘mil tlillrnctioll
`Ilmximu nieasured from native protein
`crystals and from crystals containing
`eneli of
`tliree tliilerent
`In-uvy ntuni.-5.
`In I965, after the (lIt‘\'t-!l(!pI't‘lt‘t‘tI ul lt'lU1'l.'
`I:lli(.’lr-ul
`Imalhntl.-i nl
`ll!t3ilSlll‘L‘tl’lt'fll amtl
`
`I;'nJ1IptIl:1litm,
`
`till
`
`itnalgi: was e:rlcul:1t('rl
`
`‘sync molten-ule lulfills its lum-lion wl-nan it
`l-leaves the mlmlu-ale hetwen.-II the D and the
`E ring. Note the distortion oi the D ring.
`vI'l‘Iit‘h [rushes four at its alums irlln ll l‘|lIIl‘IB..
`
`
`
`.'— .7. -5-1-
`
`mm the lmsis of nearly 10,000 diffrac-
`tion |'I'lilXlll'|i1, which resnlvetl
`features
`
`st-painited by two angstrnms. Apart from
`slmwing it
`few well-sepairateil ehleritle
`inns. whit-.h are pre.-;.ent
`l)E(.‘t11lSe
`the
`|_\-'snz'I,'t11e is cI'yst':|Ilize(l from a 5.-ulntiun
`('()I1l‘£llJIlIIg smlium chloride.
`the two-
`zmg.-atrmn image still does nut slmw in-
`lll\r'ltlttlll
`tll‘l}|YI.‘i as sepuriItr.'
`rnaxilmi
`in
`the eletstmn-density map. The level of
`resolution is high enough. lmwever. for
`llIit.tl_\' til" the gl't:IlI])5 at atoms in he ::le:u'-
`ly 1‘t?Ct}gl|t'/.'ctl)l{3.
`
`The l.ysu2_ynn- .\’lnlecnl:-
`
`:tpp4=.urs
`The main pnlylieptidt-' £.‘lt:tiII
`us :1 euntinnuus riblmn of L-leetrun deu-
`
`sily running through the image with
`regnlm-l_v spsievcl prlmiontories an it that
`art‘ clmructeristir.- of the eurliunyl grcmps
`[(C0)
`that
`l'lIiI.l‘l£ catch peplille lmnI.'l.
`In same regions the chain is folded in
`\\=:i}'s that am:
`lumiliair from theoretical
`stndie.-a ui polypeptide mnfigurntlnns
`and from the stnlctllre nriulyses nf lTl.}»‘II-
`gluliiu and fibrmis proteins such as the.
`kemtin nf hair. The nmirlu acid residues
`
`in l_V.\'t)Z_\-'i‘lI|.“' have new been designated
`lay nnmh-er;
`the residues numheretl 5
`tlllmigll
`I5. 2-]
`tliruugli
`3-1 and 38
`lhrnugh 98 form three lengths til‘ "nlplm
`helix." the r.-nnl'nrn1utitm that was pro-
`Imst-tl by Llnns Pxuiling auul Iinberl ll.
`(inrey in 15151 uml
`that was fnmnl by
`Keutlrew and his mlleagiles in be the
`must cmmnmi :n‘nmgen1ent of the clmin
`in myuglnhin. The luelixes in lystirynie,
`lmwL-\-'er_ :1ppt':I.i'
`in he .-:mI1e\.\'lI:il
`rlis-
`torted [mm the "classical"
`ftirm,
`in
`wliicll
`lnln.‘ :It0ms letlrlirm, oxygen. lil-
`trtigicti amtl
`liyrlrrignril
`tit tftl(.‘l1 peptide
`grniip lie in n pluiie that is parallel
`tn
`the axis of llie ullftltal helix. ln the l}'su-
`zyiiie Iiinlet.-nle the peptide groups in
`the helical sections tend in be rntatetl
`
`their C0
`in such at way that
`sligl|tl_\'
`gr'ulI[1.\' paint
`rnltwzml
`frum the helix
`uses ;I.lILl
`their imino gruups (NHI in-
`ward.
`
`The aunnunt of mtatiun \':tI'l<3S. heing
`slight in the helix Farmed by residues 5
`thmugh [5 mid eunsltlenthle in the one
`lUl'T't‘l£'.(l by residues 24 thrnngh 84. The
`eifeel
`til the rutntirm is that each NI-I
`
`grnup |.lI‘.Jt'.S not paint Llirr.-L-tl_\' al the CO
`grmi [1 him’ residues Imck along the chain
`but points
`instead between the (20
`groups of the residues three and four
`hack. When the NH group paints di-
`r£'ctl_\' at
`the C0 group four residues
`lirwli. as it
`(lines in the cltI.ssiL':'1l alpha
`helix. it lnrms with the CO grnnp it hy-
`llrngen bond {the weal: cliemit-all bond
`in wliich at
`liytlrugen ntnm nets as it
`
`hridge). In the lysrizyane helises the hy-
`drogen bond is iurilied snmewliere he-
`t\-.*een two CO groups. giving rise to :1
`structure llIlE|'l‘rIE(ll&ltE hetween that iii
`
`an alpha helix and that til’ it more sym-
`metrical helix with :: L|rree-lald s_vinme—
`try axis that was discussetl by Lawrence
`llmgg, Kemlrew imtl Plarttlz in 1950.
`There is a further short length ol helix
`lresitlnes BU tlimtlgh B5)
`in which the
`hytlrrigerl-llmiding urmrigemeiit is quite
`Clllfie in than in the three-Fuld helix. tlllll
`also an isulntetl
`tum {resitlnes 119
`thruugh 122] of
`three-telcl helix. Fur-
`thennnre, the peptide at the far end of
`helix 5 thmngh I5 is in the t.-unfnrmau
`tinn til the three-frilcl helix. and the hy-
`drtigeu bi‘.-ml lrom its NH guilt]: is nnitle
`ii) the C0 three residues ban.-l< rather
`than l'mn'.
`
`Partly lice’-uuse at these irregnliwitit-s
`in the structure of Iysuzyiiie.
`the pre-
`portion of its polypcptitle cluiin in the
`ailplia-helix (‘tItIl'DI'Tl]tlfifJn is difficult
`to
`mlciilate in in meaningful way inr ::m'n-
`parisnn with the f.-stiniates nlntainetl by
`other
`Inetl1mls.'|n1l
`it
`is clearly less
`than lmll
`the prupnrlitm 0l)ser\'e(l
`in
`rnynglnlnili,
`in which helical
`regitms
`mnlze up ttlltltll 75 percent til the clmin.
`Tlic lysrizyziie iimlecirle dues int.-Imle,
`lmwever, an example of iunither regu-
`lar cnnlnnnntinn pi‘(.‘.LllI:l.e'(l
`lay Pauling
`and Corey. This
`is
`the "amtipnrnllel
`pleatetl slleet." wliielu is lieliuvetl to be
`the.
`limit: striietnre of the fihmus pro-
`tein silk nnrl in which, as the l‘.Ii|lflE' sug-
`gests, two lengths ni pulypeptitle chain
`run p:u-nllel
`tu eat.-I1 other in opposite
`tll'f'L‘L'litJ1‘IS. This .~:truetu1'e again is stabi-
`lized hy hydrogen lmntls between the
`NH tIlI(.l CO gI't)np.-' nl the nlitin chain.
`Resirhles -‘ll through 45 and 50 through
`5~l in the l_v.'tu'(.yII1e umlecule lurm .‘iltt'l'l
`1| slnlctnre. with the e(mm‘.L-tillg resi-
`dues 46 tlirmlgli -19 Folded into :1 hair-
`pin bend l1et\veen the twn lengths nl
`c-aiiipirmtively exrencled chain. The re-
`Ilnlimler of
`the pnlypeptitle chain is
`ftiltled in irregular \v:1_vs
`llnlt
`lnrve nu
`simple shnrt dest-riptimi.
`liven tlmngh the level uf resnlntiun
`:r(-hievetl in mar present image was not
`ennngli
`to rt-.-salve
`iridivitluul
`alllltllri.
`nlnn}-'
`til
`the stile t.‘l‘Iaii1s
`('h:IrtIL'l.‘e.rl.‘rlic'
`ul the .-nniuu i1L'l(l residues were reaulily
`identifiable from their general slmpe.
`The
`tour disnlfide bfitlgcs.
`for
`ex-
`mnple, are llI‘.|1“l\I:'ll hy slmrt rud.-5 of high
`eletrlrun dr,-nsil_v t..-urreslriunding tn the
`lwn rt-.lutivel_v LIL-use sulfur n.tcm1s with-
`in llltfltl. The six lryptopllun residues
`ulsn were easily 1'er.-ngriizetl by the ex-
`Iemletl electron density pl‘lKl1lt.'t't.l hy
`lllt.‘ lnrgc tlnulile-ring st1‘I.It:t11!'t.-s in their
`
`85
`
`

`

`liquid. Such “polar” side chains are
`l1ydrophilic—att:racted to water;
`they
`are found in aspartic acid and glutanile
`acid residues and in lysine, argtnine
`and histidine residues, which have basic
`side groups. On the other hand, most
`of the Inatlcetlly uonpolar and hydro-
`phobic side clmius (for example those
`found in leucinc and isolcucine resi~
`
`dues) are shielded from the surrotuniing
`liquid by more polar parts of the mole-
`cule. In fact. as was predicted by Sir
`Erie Rideel {who was at one time direc-
`tor ol the Royal Institution) and Irving
`Langmuir. lysozyme. like myoglobin, is
`quite well described as an oil drop with
`a polar coat. Herc it
`is important
`to
`note that the envirotunent of each mole-
`
`cule. in the crystalline state is not signifi-
`cantly cliiferent from its natural environ-
`ment
`in the living cell. The crystals
`tliemzielves include :1
`large proportion
`{some 35 percent by weight) of mostly
`watery liquid of crystallization. The
`effect of the surrounding liquid on the
`protein colifonnation thus is llliely to be
`rrulch the same in the crystals as it is in
`solution.
`
`the observed
`that
`then,
`It appears,
`conformation is preferred because in it
`the hydrophobic side chains are kept
`out of contact with the surrounding
`liquid whereas the polar side chains
`are generally exposed to it. In this way
`the system consisting of
`the protein
`and the solvent attains a minimum free
`
`energy, partly because of the large num-
`ber of
`favorable interactions of
`like
`
`groups witl'u'n the protein molecule and
`between it and the surrounding liquid,
`ant! partly because of
`lhe relatively
`high disorder of the water molecules
`that are in contact only with other
`polar groups of atoms.
`generalizntiotls,
`Guided
`by
`the.-ie
`many workers are now interested in the
`possibility of predicting the conforma-
`
`FIRST 56 Rfigl DUES in lyaozytne molecule contain a higher proportion ol symmetrically
`organized regions than does all the rest of the molecule. Residues 5 through 15 and 24
`through 34 (right) form two regions in which hydrogen bonds (gray) hold the residues in
`a helical configuration close to that of the "classical" alpha helix. Residues -Il through 45
`and 50 through 5-! {left} fold back against each other to term a “pleated sheet.“ also held
`together by hydrogen bonds. In addition the hydrogen bond lnetween residues 1 and 40
`ties the limit 40 residues into a compact structure that may have been lolcled in this way
`hclore the molecule was fully syntlreeiaccl {see illustration at the bottom of these two pages}.
`
`side chains. Many of the other residues
`also were easily identifiable, but it was
`new-a-tireless most
`important
`for
`the
`rapid and reliable interpretation of the
`image that the results of the chemical
`analysis were already available. ‘Nitlt
`their help more than 95 percent of the
`atoms
`in the molecule were readily
`identified and located within about .25
`angslront.
`Further efforts at improving the ac-
`cnntcy with which the atoms have been
`located is in progress, but an almost
`complete description of
`the lysozynte
`umlc<:ule now exists [sec illustration on
`pages 80 and 81]. By studying it and
`
`the results of some further experiments
`we can begin to suggest answers to two
`importsmt questions: How does :1 mole-
`cule such as this one attain its observed
`ctmfonnafion? How does it function as
`
`an enzyme, or biological catalyst?
`Inspection of the lysozyrne molecule
`immediately suggests two generaliza-
`tions about its conforrnalion that agree
`well with those arrived at earlier in the
`
`study of myoglobiu. It is oiavious that
`certain residues with acidic and basic
`side chains that
`ionize, or dissociate,
`on Contact with water are all on the
`surface of
`the molecule more or less
`
`readily accessible to the surrounding
`
`GROWING. POLYPEPTIDE CHAIN
`
`RIBOSDME
`
`MESSFNGER RNA
`
`CO DON N UM BER 1
`
`FOLDING OF PROTEIN MOLECULE may talte place as the grow-
`ing polypeptide rlmin is being synthesized by the intracellular par-
`ticles called rilnoaornes. The genetic mt-as-age specilyiog the amino
`
`ocid sequence of each protein is coded in “messenger” ribonucleic
`acid (RNAI. It is believed Ieaeral ribosomes travel simultaneously
`along this long-chain molecule. reading the message as they 50.
`
`36
`
`‘it
`
`Page 10
`
`

`

`I
`
`I41:
`
`L.
`
`1.__
`
`tion of a protein molecule from its
`chemical formula alone [see "Molecular
`Model-htfiltling by Computer." by Cy-
`rus Levialhal; SClEl\’TIFl(:
`z\MI:‘.H1c;A:~:.
`
`juuel. The task of exploring all possible
`coalonnatlons in the search for the one
`
`of lowest free energy seems likely. how-
`ever, to remain beyond the power of any
`imaginalalc computer. On a conservative
`estimate it would he necessary to con-
`sitler some Illl‘-3' t.liIlt=.rcI|t L‘(‘JT)fDl‘l1'|‘.'ll‘l()l1S
`
`lor the 'lysoz_vn1e molecule in any gen-
`eral search lor the one with lninimuan
`
`Free energy. Since this numlaer is far
`greater l'.la-m the number of particles in
`tho observalilc lII1ivcrse, it is clear that
`slrnplifying asslrtnptitarns will liave to he
`made il (.‘:ll(.'I.tl£l.l.l0ll5 of this kind are to
`succeed.
`
`The Folding of Lysozyrne
`
`For some time Peter Dunnill and I
`
`lmvc been trying to dc-.-velop :1 model of
`prntein-folding that promises to malce
`practicable cult:nlations of the IIIlIllIlIl11TI
`energy ('unfom1atio11 and that is. at the
`some time. qaalitatis-el_v consistent with
`the olaserved structure of myoglohin
`and lysozymc. This model makes use
`of our present
`lmowlctlge of the \.-my
`in which proteins are sjrntllesized in
`the living cell. For example.
`it
`is well
`known, from experiments by Howard -.\-I.
`Dintzis it.J'l(l. by Christian ll. Anfinsen
`and Robert Cautlield. that protein mole-
`cules are synthesized from the terminal
`amino end of their polypeptide chain.
`The nature of
`the synthetic mecha-
`nism, which involves the intracellular
`partit-les called ribosomes working in
`collaboration with two forms of ribonu-
`
`cleic acid ("messenger" RNA and "trans-
`fer" RNA),
`is increasingly well under-
`stood in principle. although the detailed
`environment of
`the growing protein
`chain remains Iulluuown. Nevertheless,
`
`it seems as reasonable as-au111ption that,
`as the synthesis proceeds,
`the amino
`end of the chain becomes separated by
`an increasing (lislance from the point. ol
`attachment to the ribosome, and that the
`
`folding of the protein chain to its no-
`tive conformation begins at
`this end
`ct-on |)el'orc the 5_Vlll.ll&.SlS is complete.
`Avumliug to our present
`ideas. parts
`of the polypepticlne cllain. parrit-ularl_v
`those near the terminal amino end. may
`fold into stable L't'Jnl(:r1n:ttio11s that can
`
`still be recognized in the linishecl mole-
`cule and that act as "illtt-nlal tcinplatcs,”
`or centers. around which the rest of
`the chain is folded [sec illrt-rtrofion oi
`lwflom of Hmsr!
`two pages].
`It may
`therefore be useful to look for the stable
`
`conformations of parts of the polypep—
`tide chain and to avoid studying all the
`possible conformations of
`the whole
`tnolctrule.
`
`Inspection of the Iysozymo molecule
`provirlcs qualitative support for these
`ideas [see top illrtstrolitlli on rrppositc
`ptrgcl. The first
`-10 residues from the
`terniinal amino end fo11'r1
`:1 mmpxlct
`structure [residues 1 and 40 are linked
`by a hyclrogen bond) with a hydropho-
`bic interior and a relatively hydrophilic
`surluce that seems likely to laws been
`folded in this way. or in :1 simply re»
`lated way. before the molecule was
`fully syntliesized. It may also be im-
`portant
`to observe that
`this part of
`the molecule includes more alpha lielix
`than the remainder does.
`These first 40 residues
`
`include a
`
`mixture ol hydrophobic and hydro-
`philic side clmins, but the next 14 resi-
`dues in the sequence are all h}'d1'op|1ilic:
`it is interesting, and p=ossihl_\r' significant.
`that these are the residues in the anti-
`
`pnrallel pleated sheet, which lies out
`of contact with the globular subn1olc-
`cule formed by the earlier residues. In
`the light of our model of protein fold-
`
`that
`speculation is
`ing the obvious
`there is no incentive to fold these by-
`drophilic residues in contact with the
`lirst part of
`the chain until
`the hy-
`drophobic residues 55 (isoleucinel and
`53 {lent-inc) have to be shielded from
`
`Contact with the surrounding liquid.
`It seems
`reasonable to suppose that
`at
`this stage residues 41
`through 54
`fold back on tllelnsclves,
`lornling the
`pleated-shoot strut-ture and lnn'_ving the
`hydrophobic side chains in the initial
`hydrophobic pocket.
`Similar considerations appear to gov-
`cm the folding of the rest of the mole-
`cule.
`In brief. residues 57 through 86
`are Folded in contact with the 11|eal'ed-
`sheet structure so that at
`this stage
`of the pro:-ess—if indeed it follows this
`L-ourse—tl1c folded chain forms a struc-
`
`ture with two wings lying at an angle
`to each other.
`llcsirlaes 88 through 96
`lonn a length of alpha helix. one side
`of \\-"l1l(.'l1 is pretlorninantly hydrophobic.
`because of an appropriate alternation
`of polar and nonpolar residtics in that
`part of the sequence. This helis lies in
`the gap between the two wings formed
`hy the earlier resitlues, with its hydro-
`phobic side l)l.1l‘if‘.'Ll Within the molecule.
`The gap between the two wings is not
`completely filled
`the helix, however;
`it is transformed into a deep cleft mn-
`ning up one side of the molecule. As we
`slmll see. this cleft forms the active site
`of the em:yu1e. The remuilling residtles
`are folded around the globular unit
`formed by the terminal amino and of
`the polypeptide Ichain.
`This model of protcin—l'oldi11g call he
`tested in a number of ways. for example
`by stlxdying the conlonoatiou of
`the
`lirst 40 resitlilcs in isolation both di-
`
`
`
`Presnrnably the messenger RNA for lysozyme contains 129 “ro-
`dent.” one for each amino acid. Amino acids are delivered to the
`site of synrlheeis by molecules of “transfer” RNA (dark color]. The
`
`illmilrnlion shows how the lysoayme chain would lengthen as a ri-
`hosorne travel: along the messenger RNA molecule. Here. hypothal-
`irally. the polypeptide is shown folding directly into its final shape.
`
`
`
`

`

`rcctly (after removal of the rest of the
`molecule] and by computation. Ulti-
`mately, of course, the model will be re-
`garded as satisfactory only if it helps us
`to predict how other protein molecules
`are folded from a knowletlge of their
`chcniicul structure alone.
`
`The Aclivitgr of Lysozymv
`
`