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Kinetics of tryptic hydrolysis as a probe of the structure of human plasma apolipoprotein A-II
P2sevicr
Kinetics of tryptic hydrolysis as a probe of the structure of human p]a.¢ma apolipoprotein A-It John B, Massey, Diane LM. Hickson-Bick, Antonio M. Gotto, I1-.and Henry J. PownaH (R~v~,t 9 Ma~ 1989)
Keywards: Ap.glipopr~teit~A-It: Slrtmeu~.~'mletiu'~zelot~um~hip;"]'ryp4]chydrolssLs:(HLtmanp]a~nml A~ n model system to t m ~ alp0111mp¢oteinsirlt~tlu~d-~,~ric~,Ic,. ~, :'2q41their relatio~nsbips to Izroteolyfie event, i, the klnetl~ M tr~p4~ hytlr~ysis o f apolIpoprotetn A-JI ~apo A d D yeas i~estigated in solution anti. in ae,_,,~iati~ wlflt #hos#bo]ipid. "l'be raUs o f apt0earastce and i~ulifies o f spedfie pepddes .a,~e determined hy r * v e r ~ - p l m ~ hlgh-pe~fonmttme liquid e ] m ~ u m t o ~ , rand imago i c i d mn~]y,M~ reslu¢.edvely. F~r the kl~elle~ of h~'dpul),~ of ~,po A - [ i in solution, the e~rboxyl-termil~d i~Plides o f rasidl~es :~-7"7 and :56-7"7 apl~ flr~t, fe~lowedl hy Pel#~k"~ M' tesidhle~ 4 - 2 3 , 29-,~, 40-44 and 4S-,5,4, whid~ appe#*x'd at ncm'[y itlentieal rates~ [ ' h e ~.ioetles o f hydrolysis of apo ~ - I I m.soel~ted ,a~lb l.~dlmys[stoy]-,m'~yc'eeo-3~phosp~to¢'boline showed se'a2,m]difl'erenc-e~. Rr~l~ a lO0-I,n~lI ~ anmunt ~r ID,~in was m~edle~to oblaln a ~,imilar rate o f pt~dm-t I,mmatlno; m~cem~ a new p * q ~ appeared, elming earlier t-÷lan A - l [ ~ hm4ng tl similar llmlno aeld ¢omposl/1011;mid IIMrtLth~ mlallve hulas of aplpearmlce ot pepfides were d~'fe~_..~L ~ ~ ~'tlel'll~ Sllff~ll~illl~ ~ gontls SUL~eptil~eto Iryl~in ¢'teavage was deletmined I~- several p¢~lK'llw uJ~mridmm. The lyshie amino ~ i d bonds were folmd I~ I ~ in reglolt~ define~ by a hi~g heli¢'al mlll~ipmthic The *~luced ms~q~'bili*y to tTyp4ic by~rol~sis of sl~-II ~ i ~ * t ~ with phmsplmlip~i epp~m,s to be ~ ~o a f~ eveerl~ o f StaI~IIIzItt~OtlM I~'~ein ~oJOelmy Stlltetm-e. As. a ~ e ~ n ~ e . , the ]ysi~e amino acid I~nds are in fo~ded I ~ n s n l ~ e I~v~M~n wince tl~s'y are confornm~ons]ly ira"Ida m enxym~ie hydroid, is. 9 y xle~e o f ~ r e . p c e d i c f i o n methods, it is p4x~ihle to designate ~hieh re#ort~ of apolipopcmeins may' be important in prote&V~is,
L-nrodtmfion The prott.';,n components o[ plasma lipoproteins, termed apolipoprow.lns, distribute diffe~emly among the lipoproleins of different de.~ity classe~, gave di[fe~nt primary structures, and ~ntribute differently to the control of lipid metabohsm as activators of lipid, catabolizing taazymc:sand a.s ligands for receptor.attila, ted uptake [3_].Apolipoproteins have different metal0oliu fun~.'tloas, but ~h~r~ a common Mru~.-tutai feature; they associate with a phosphollpld/w~t~r int~f~o:, F~am analysis of tlu: primary st.tuetur¢, of apolipo,prtRei~s, a
Abbyt~L~tim~; HI~'L hbgh,d~m,~lYhl~Pe~l*ln~; ~lp~A.I, ~llcKtllpr~Oolean A-l; apo A-[l, apolipoproton A-II: UNIt'. ].Z-clLTn~nstoyt-zrnsl_',nxT~-3~,hco3~myldx~m;]IPLC,~gh-perl'mama~oellClUld~hrom~tlo~taph$:~v/s,v,",~,ighl/w¢ight:VH.S¢1i¢*1~mrp~i~hi¢moment; (SIN ltyd~patM: ( ~ ) , I~li¢.alprcbsl'dli~y: Odm-HC1.guanldi~¢ b~,d,~id¢, cm~pon6¢n~.e:J,B,Mat,~y.l.~-pa~mrn~,of MedKil~."l'h¢M~hcKIia H~lal, 6.~i~Fatwdn,M-S,A~¢OI.Houu~[011,3-X7703~,.U,$,A,
general model o f these lipid.associating domains ba.~d on tn~ concept o f an ampt~pathi~ helix was proposed [2,3], Apo-B-IO0 may bc an c'~t_'cpti,bn, hc*.:aus¢ ~;trucrural ana]ysls Lndicat*s th~1 h contains more ~-sheet st~cture than the other apolipoprotcms [41. Even with mode]s for ]ipid-proteln association, the overall slruutural organization of indivldual a p ~ i p o p ~ t~ns on the surf.ace of a lipoprotein is not known, Studies using chemical labeling, immunological meth(~ls. and proteolysts h~ve dctnonMr#.ed float epo]ipt~protr.,irrs are on the fipoprot6n sur~ra~ [.%6]. tt~hlnLiquesar~ ~.s~ulacd to diff~entiatc betwe,~t Jcg~oas that are accessible to the t~:agent ('exposed'). and regions tha r. are non-acce&~ihlo ('buried'). Correlations of these studies with the known s~que..n~¢ and p~edicted stru~.trite5 of apotlpoprot¢ins ka-~¢ h ~ n aAtb~x:~oas in such heterogeneous systems a:s native high.density lipoproteln~ which contain sevc'~a| apolipopmtcJ.ns. Mor,~2mt:r, LIt~sT~t:ili¢ Set;lu~UOC:r~gions ¢.g., antig~nlc site~, prot~lyuc cleavage sites, ctc,, of ti'w apoliproteins ~.hich am experimentally proceed have not been identified. Con~llcting reports exist concerning the extent of tryptic
1~1~7.4~.'~g/$9/.~:1.$0~: Lgg9El~ier ~:icm~*rpla0kis~h~T~B,V.ClNomedneLdD)VLb3OL3)
hydmlysi.~ of I'tDL and a p ~ H D L using sohiblc and immobilized trypsin [5]. Most studies on native H D L have indicated that a?o A-l| i~, more su~eptibie to praleolytic cleav~e than apo A-I [5,7]. Addltincally, self-as~ialion of the apeprotein ix a variable, tnvoIvemeat of pmtcolysis in "~ipol~Ol~in mclaboli~m has been demonstrated Jn the co- and post-[raoslatioaal modiGcation of the apoiipo~oteins [6÷8] and in the d e a w g e of mature apelipepre,mms on the lipopmtein panicle 17,9], To develop p~oteo~ysisas a probe of the stmctef¢ of apolipoprmeins, w e sludied the kinetics o1" tfyptic byd ~ y ~ s of apo A-II in solutiOn and comple,ted with pho~f, h01ipid~. |'he rate of appeara~e and identity of specific peptldes w e ~ delermilu~d by r~,~.rs~l.phase HPLC anti amino acM analysis, respectively. Predictive algonthms of halieal probability [10}, hydrop~thy i ] l j end helical amphipethic moment [12~14] have beam to aou|yee the ~ a n d a r y stroctare surroufid~,l~ the bonds sosceptible to tryptic cleavage. Although its runetiOns are uncertain, human apo A-It is or inlerest because it is a s.ma]t protein oonsi~fing of two id¢oti~l polypeptid¢ chains of 77 amino acids that ate linked by a single disullide bond at r e v u e (, {15}, F u r t h e ~ e . in ,dtro studies have sugi~'sted that apO A-II may regulate the activities o( [ecithio-clso]4=s~=xol acyhraasferese [16[ and hepatic |ipase [t7-191. The .~vanta~s of using apo A.]] an~ ~ a t its structure has been extensively studied w~th native and syfitl~ie peptide fragments 120-22] and that the stn~etures o[ apo A-]l/phospheliQ~d o3mplexes are well chafacteri~xl [32-251. Expednt~*~ l=,~ed~'es M~teria/s All materials and rnethc~dsfor the preparation of apo A-ll and D M P C / a p o A-II r~.ombinants ha'~e been described [23-25]. For this study, a D M P C / a p o A-l| r¢~n3binant with a lipid/psotc~n molar st~ichiometry of 7S:I was u.~d. Trypsin-TPCK (226 units/toni was from Worthington Bk~hemicals (Freehold, N J), All o t h ~ salts and solveal$ w ~ from comme¢~al ,,,~ndnrs. A sta~dar~ buffer composed or 10 mM T~is, 1(~, mM NaCt. ] m M sodium azide. I mM EDTA. 10 mM CaCI2 I'p|] 7,4) wax used throughout, Metho~ Tryptic pcpLidcs of apo A-]] w~r¢ isolated on a Spectra Phyr-~, ~O00-B high.pedmmance tlquJd thenmatog~aph with an SP-8400 variable wavelength dametar, using a C, mversed.pl~lse ,~ynchropa~h RP4 colamn ( 2 ~ x 4.1 ram). proteins and peptides were dated with a linca~ ~edient of isopropanot in ;5 mM ammonium phosphate (pH "7,3) and monitored by abs o r b a ~ at 222 rim. 1"he individual pcplides were iso. lated and i ~ t i f k ~ d by amino acid analysis,
To estimare a rate. the time for.~'~ disappearance at" the apo A . I I peak or ~tppearanee of the peptide peak wa_~ use~ a~ the ha]hime of hydrolysis (Q::). The rz/2 valt~es ~ere date.(mined by ~sual ins~liO.n of the data_ The rate of appearance of s p ~ f i c apo A-II p e p t i d ~ was de,ermined as a far~.tion of the incubation time with o3,psin. An aliquot of the sample mixtn~ containinn ~,~,sin wax collected at fegtdas iater~aL~ and quenched w~.th 6 M guanidine.HC1 a n d the~ rapidly frozen ia an ethanol/~olid CO= bath, The samples w¢]~ stot'cd frozen until analyzed by ~versed-phas¢ HPLC, Each peptid¢ was quantified, by zr~asuring the peak h~gh~_and ¢orapar~ng it to the peak height after total enzymatic hydrolysis. The int~a'ated axens of all the l~aks wese identical for each H P L C analysis, indicating the repr4~ucibility of ~ t i d e isolation on the column. To follow the ktheti¢~, the rates of c'ii~appearance o( A-II and the appearance of tryptlc peptides, residues 4-23. 29-39. 40-44, 43= 54, 55-77 a n d 56-77, were ~asure, d. Citt:alaF ~ichrGic ~ , ~ t r a were mea~tlred on a Jas, eo J-51]OA spectropolarimcter [25j and f l a o ~ r y , : e speclr~_ wine measured on a S L M 8000 spectrophotom¢4er (SLM Instrument% Urbana, IL). Thermal d~aturation of apo A . I [ / D M P C r~ombin~n~ was ]'olh~wed by Chang~ in the flaotescence intensity of sl..e ry~osir~e re'dues (etnisaion wavelength was 320 nm and excitation wa~le~g~h was 275 am) and in the molar dfipt{city me..asore~ at 222 nm ([6']~2~), The tempexattzre dependence of the speetroseopie measumm~ts was achieved by placing the thermal oouple of a Bailey Inatturnents digital thermometer (Modcl Bat ~) directly in tlre 1;uvet~ autd recording the spectral intensity vs. temperatu~. The thermos=ted sample cells w e ~ initially cooled to pr0x. 0eC by a Br;.nkman ¢i~culating. water ba~.h and the heatin8 cur~ initiating by ch~mging the thermostaq on the water bath to 100'~C which occurred OVer about 2 h. The equilibrium COnstants and standard &ee energies (.,4G°) o[ dcoaturatlon fat apo A - I I / D M P C combir~ants were calculated rron~ ~rvcs of tyrosine fluore~ence or [g]=z= vs, lamp.ratine, assuming thai the denaturation was a reversible two-s~te pr~s,~ as r,,esib©d by Rcijnoud and Phillips 1261. The secondary str~ture of a ~ A-[I was analyzed hy sa,~eral pro:|ictive algorithms which included u~t¢ helical hydrophobia momem algorithm of Eisenherg et al. q12-14]~ the hydropathy tr~hod of Kytc and Doolittle [1.l], and the helical pm~.bility as dey~ribed by, Chou aL11dFasm,an []0]. For a hydrophobicity ~al©, the .scale of L,fviu (27}, which ia based in pm't ¢~ the partitionir~g of m-nino acids between water and an organic phase E2gl, was ased, This u;ale correlates well with the buryin 8 or a Itsidue in a hydro|phobic en'.Amnm,~t j29]. A 'moving segment' of deven residues was uu~d to calculate the helical hydrophobia moment, hydmpathy ned helical probability aioutld a specific resldoe. The aver-
I~educe~P
n~.~ A-]I 5 0
I
--
-
-
-
-
~5 o
rqn~-44
,
~,
40
6o
~ ~
80
FIB. l, Tr'jpl]¢ f r e t s I7[ elpi>A - I I " ~ unatslcd by I¢~¢w-,cd-plllL~,C HPLC.~ypically, ~ 0 p j t 0 f Wocemin 1'00 p l o t I~,J/i'=l ~ontalnlng } M ~rdflt-HCI IVC~lre ~llal~e'l~ ~e!', T ~ pt~ljr~s 3~¢~c ¢1LI1¢~ " ~ l ] l a linear ~adiet~t of isulfnr I p H ?,3l al ~ ~ow tale # 0,~ ml/t~Jn, The prmein peeks were colin:led al~l idenlil~ecl by amino acid ana]w;~ T h e appcoc~zt¢ X~l~enc~ [or ocsidu¢~ 4-23, 24-2~, 29-39, 31-39, ,':. ,~,. 4~-34. ~ 5 - ~ ,end 56-77 "4vet¢ idellli(ied, T'~e nystei~t¢.~oacaiuing pepllde wa~.rurgher ,,~nhcd ~Cl~n wilh dilhioIhr~to~ bel'(~ I ~ ' ILPLC ~nal)'si~_ "i~m~redu<~!d pel~d¢ wa~ shif!ed m b~.~me ,duu:d at a ~ = r p¢l¢Cnl~3~g¢O1
-
~ ~ 5 ~ "< ~ 10 " i~
o
..~
m,/X2
~ "F
5 • 0 0
Z
4
~
i~1~an~,
2
4
I~
~
11~. h }rig.,2. Th¢ kLn=tJc~of t.~'I~r0n hydnalygi~canapo A-El ,n ~,Mugio~wnt¢ tolt~ by [be dl:;3p~:atr#.~.J~ O1"~.~f7A-t1 {A. I~ arud Ebb"~l~l~arance
age values for 1he whole prolein were calnulated ~ the average Of the tot0.1 'TY~ovirlg sgglllcnts' p ~ ' residue for
,~f ~ e r a l IT3C~tic~pnpLide~ c~r:~ponding m r~idans ~6-T? [A. @L
the p~ot~in s e q u e n c e ,
55 .?'7 {B). 2 9 . . ~ IC.~ ~ - 2 3 (D). 4c-J,4 iE~ a ~ l ~5-_'.4 IF)- T o p~rioem Ih~" ¢~n~,~i~ml,~nl~,IIl~J]li!P~cbampl~ o4rapo-il 2~ 37~c n ~ l a i ~ -
~'qtl~l~i T h e separation of the tryptin p~ptid~s wcJ~ identified
l ) 0.r~d comparison
peptides o f a l ~
by amino
A-]I
a c i d aoaly~is ( T a b l e
to the known ~.iucnt~,
T h e di-
l O O m M NaLI. I mM EDTA. I rnM sodio.rnaz~de, l~mM CaCi~ t p H 7.45 were added to 2? ~1 of a 50:1 dilu[ic~ o] a s[ock trypsin ~olullon ¢0.8 mg/ml 3 in 0,00l ~-t HCL A[ lh¢ end g,f e:w:h ,tree ia~erv~. 6 M G d m - I I C I was ~,¢,'d It3 sl¢~Dthe rP~ction ~;~ the mLxlu~ w,~s qu;@,b' - ?1~° C I'r~'¢¢r and L I l ~ j r l ~o :~ul~mbl¢nt InmpuraLuL-¢ L~cfor¢-in3¢don ingO Iht¢ H['LC. 12unb.thin, Lion 1200 ~,l)con
TABLE I .4min,~ acid c~mpr*,~i,'.'on Amino J]¢id
r]/ re_Vphr, pe'W~.~ 4~f =,m~ A IL "
T~'D[iC ~ L ~ 4 -t:~l
~4-~g
Cy~ ~ A,p Thrb
- fl) l . t {11 - 4`25
LL8 ~1~
Glu Pro Gly Ala Val Met Ik L~u Tyr Phc Lys
,i.g {4] l j ) (15 1.3 (15
|.2 I1}
2.7 (31
2 9 - ~,9
31-. 39
J.2 qS~ I.I II}
-~.5 t3] 1,~) ( I )
t.8 IZ} 1.0 I1}
1.9 ~Z)
t.~3 (l)
1.0 (I}
40-44
I . ~ (l~
34- f',4
55-.77
f~5- 7T
0.7~il
l.I (l) - t3j
1.1 ( i } - I3]
4.2{41 1,0(ll ~, I (?]
4.2 (4} L.0{b 2,2 (.l}
2.442~ L.~it>
~ (I (25
L0(Z)
2.04:2]
2.~(2)
3.213] ~J.l~(1) ~'.0 q21 1,1 (15
3.1 q31 I,~ (l) 2-'f,I4`'~1
15.5{])
C'.9 Ii~ 1,~ 4`1) 1.7 (2) IJ~ (1) 1.6 (11
1X}(I}
0,~O)
1.8(25
1.r) 4`1}
2.0 (2~ 0.7 (l) 0l~ Ill 1.0 t l )
1.9(2)
• Fo~ amino ~ i d ar~ly~[~, 5 n n ~ l n[ tryp~ini:,~d ap.~ A - I I ~ u~'~ to L~I~U~ t k p~pii~:~ 1~" rm~cl-ph~L~C HPLC. T I ~ p~aks mca~ur~l at 220 nm ~ n c i ~ a t ~ l and s u b ~ t~ hydro])~ (24 h. 110eC) ifl ~ldc], Cv,n~uP*l¢i~[0b~. em{doyin~L 6 M HCE A~aly~is ~ pcH-orm~d un au LKB a11~11oach]5 anal~LL~r. Tla¢ valuta: rc~l'~-~lt Lhe a'a~lrag~oF th~-~ d e t ~ r m i r ~ . t i ~ : the L,'aLLI~in T J a ~ t l l ~ w aJ~ LL'I~dlRpolrL'~[~l]vullJ~. ~"C y ~ Tiffalod ~ v a ] ~ ar~ L~34LqLlanlitaL~l~ I:'APC,~PLI~C q.hc'y~ parb~.]ly dc~tr¢ly~J in lhe a~la]~2~.
I ],I
•~.ll'id,~.con~,imn B peptitJer r~$idl,~-~; 4--23i wa~; f.J.rll'Je_r i d t n t i f l ~ by r ~ c t i o n w]lh dithiothrci~l. The kinc s or hydrolysis by t~'ypsir~, 0.0O~ (w/w) tryp.
---n
,
.
t0
0 10
,C
b I
I
.
__t
D
. . .
.
.
.
.
(Ti~ 2A) ~nd 55-77 (Fi~ 2B). After p m l o o ~ d i n c u ~ zion, Chezunc~t or peplide 5~-77 db~rc&~l ~ad that or ~-77 .~d. ~r~lJneg ~y~I~i~ ~|~-~ ~?T¢ ¢se~maced by visual Lr,.~tic~n of the d~ca. The vaJ~¢ or t,/~ for apo AIII ~uas --0..~ h. while valu~ fo~ the al~raJ~ of the pcptidcs ffesidu¢~ 56-11, g/z "~ "~ h: ~d~¢ s ~5 - ~ , I { / ~ - 0 . 8 h) w©r¢ slichtly longer. The k3eetics of appearance of peptidts 4-23 (~,/~ - 3.5 h), 29-J9 (~'t/:" 3,2: h)i ~ - - ~ (t]/~ = 3-6 h)q and 45-54 (fl~ - 3,3 h) (Fig, 2C~V) exhibited a~t initial I ~ pha,~ tmtil m ~ t of the apo A-II had disappeared ~nd ptptides 55-?7 ~ d 56-'/7 had bec~ formed. The kinetics or" tt3~pti¢ hydrolysis of apo A-]I in a D M P C / a p o A-{I recombiJtan*= (75:1, ~ l / m o l ~ indiw ~ n .e~__ ~-] lq O~l~n
a
':
,"
O
'-:'='i, o
....
.~
0
k
10
20 31) ~D?'B 10 20 T|me. I~ Pill., .'I.~ : kin~Ju ud"l~'.~l~inl',),d,'rt~)'.qis¢,I'al~ A-LI "co~mbin~ w~th ~hol~d ~ follc~,'~lI~ Pal~id¢~ ! . ~ s by HPLC,T ~ ~ l t ~
s i n ' ~ g rate of .p¢"~Li4~-J'o'r,m~t.~3"l'l
w't.S the ~Tpeasan~ of an iat¢,kml.~(¢ Ip~'pd~c. apo A.II" (Fi 8, 3B), that d u ~ d tJishtty taditr than apo A-|.. Within 4~tpefimtrtl~ ©rmr ( ± ] 0 ~ ) . Uu: amino a¢itt ¢ ~ : ~ s i t i o n of this peptidc was identir,~ ~ that ol ~po .~-[I. This t~eptide may I~ve been fofme.d by hyd~i)'.~ at Lys% A third major difference w ~ in the ce~axi-,~ rates or apl~acrce o[ ~ i d ~ lro¢ alpO A-I[ a . ~ @ ~ t ~ w i ~ pho~pltolipi~, the rate of d i ~ a r a n c e apo A-ll (11/--2 -- ~.S h) and the rates 0f ~,p~rar~ce of ~ l ~ i d ~ 2 9 - 3 9 {tl/: ~ 2,5 h)+ and 4 5 - 5 4 [,l'l/z -- 4.5 It)
tl~l: clut¢~ slJEh~l~ kfum ~po A, II. A ]OI)-lrdd "niger ~ ~' ~ J n w¢~ ngCded10 ~l.~d .c~nmpmr~ble~ o[ disapl~mm~ zpo ~11 io a ~u:c~nam ~ c¢~n~-~d ~o ape A~]] free in ~ol~on.
similar to that ~r 56-77 OL/~ -- 5 It) and 5~-'77 p©pcid~ ¢ o r t e ~ o ~ l i ~ , tO ~sidacs 4-23 (t~/z - ]3 h)
TADL If |J
I ~.0
4[
AI~ ~tL/DMPC
T ~ .~e C RC'M,A~ .%-I1 ~-at ~:ik ~C.s F'i~,id I=C~
.- 19 -7 m ~9
~
22S ~
42
TABLE TII PJ'edi¢ied ~¢t't#Cd:rU:at~furc ~4~almng fhe l/yp,~iyi.~v.~ceplih?t,: y o ~ in
correlate chAxng~ ifl. [~rOtCJ~ MTucture w i t h "m~tlsnrab]e thermod~natinc qu,:nt: %:, ~;=]chas ¢qui]ibrLum col3.stant,s
ape A-It
[36] and iatnnsic surface ~ctivili¢,~ [34]As a mo~el system Io probe apotipoproteja struclurc,.functlon, the kinetics of trypllc hydrolysis of ape A - l [ in solution and a,~'~OCiated with phc~pholipid weo~ dcl(:rmined. Differences "~t¢ fecund ~n both the r,~te and order of appearan~ of differcm pcptJdes. C o t . l g a r J s o n of the twz values and relative concentra= tines of trypsin used ,show that the ape A-II is approx. 5C~-timc,s more rcgstant to protealy~s when complcxcd with phosphollpid than when free irt solution, The rate of appearance of peptidc,s in tht: proteolysts of ape A-II in solutlon follows the order ape A-[I > 55 77 ---56-77 > 4-3'3 --=29-39 ~ 40 did ~=45-54, Ape A-IJ in solution has fiL[l¢ ordcr~:d s~ruetu~ ( - 3 5 ~
gesmues Lys,_~u ( Ly~2~-Am~4
,~H= 1.16
0,56
i.as
Lys~-"~zJ as
0.95
0.9~
LTs~-SerJ~ L~-Ser ~
O.4g 0.g3
1.14 1.16
Lys~.Sef ts Lys~.Olu 47 LySa¢-Lys ~s
LOg
0.33 0.1~ 0.73 1O,2:B 0.C06 0.036 ~a.(128
Lys'-A~as~ ApoA-tI meaJXVaJue
(kgal/mol)
(I.J~ O,(~ 0,&2 0.72
{g)
{kcal/mMI
(e=)
z,l~ Z.;I z.~. 1-(]5 l.,~s 1~4
* The helJctl~.mphjf~alhicmomcnt(icK)w,%5.c.ctleuhttedbythamech~ of E~abe~g e, al,112~141,the I~top~d~? I~/'1 ) ) wus ~;ak~r~=cd f0y
mr m¢i1*~of Kyt¢and Ooatinl¢ il ll, an~ ih¢ I'ali~l INo'oabilJ~' (
u~fl~in the ~aloalat;o0~.
rates of appearance of peptides from ago A-It in solu. tion ~ different from those of ape A-1I in a DPMC/apo A-l[ (75:1, reel/mot) recombbmnt, The f ~ ~¢vgv of proton-lipid association was determined by thermal denaturation of the protein as followed by changes in tyrosine fluol~scence or ¢i~ulmdichroism. T:~ble II is a compilation of the th¢,n3aody, namic parameters for the ape A-II/pho~pholipid complex. Ape A-II in ~otation has a low fr,.x~energy oF stabilizalion, , ~ G = - - 1 kcal/mol. The free energies of the ago A.lI/phospholipid complex wo~ on the order of - 7 to - 9 kca~/mol. The e a t h a l ~ values ~etermined by thermat denaturation e l ape A - l l ~'o]lowed by changes in tyrc~ine flnoreseeagg or e[liptichy were very s/mihr to tho~c measured by batch micro, calorimetry [24.30] fez protein folding upon ]told asset'Sarian, The seoandary structure of ape A-ll was analyzed by ~vmal pl~dietivc methods, Table l i t contains the colue.g for the helical hydrophc)hie momenk the hydropathy, and the h~c;al probabilily of ao eleven-residue segment surrounding the lysinc rc.,6.duc rccognlZed as the tryps/n substrate, Disomslon The phosphalipid.a~so¢iating domains of apolipoprotelns have been d~cribod in an amphlpathlc a-helical modal 12,3] wh¢'re the polar and non-polar residues lie on ~ i l e sides of the helix and the non.pola~ fan: of helix penetrates the lipid phase. The helical amp'mpathlc moment algoritkm [t2-14,33-35]can be us=d as a quantitative refits, meat of this m o ~ l . It hag been used to ana]ye~ s4~ondary structure and to directly
a-helix) a~d a small difference in the tree el~¢zgy (~.G - - 1 kea]/mol) (Table If) between folded and deanlured states~ which is similar to ~het for the stability of a Sing]{' a-helix [37], However. ape A-ll ex]fibits certain charaetcristi~ of globular protein. For example, two tyrosine n:mdues per chain are not e s p i e d to tha aqueous solvent, as determined by solvent per~urbatJofl difference spectra (Massy, J.B. and PownaJi, H.J., unpablJshod data), The disappearance of intact ape A-It obviously would occur firs k since only one tDgtic ¢leav0ge ~ moleeo]e is required, The Formation of pept~des 55. 77 and 5 6 . 7 7 rcqinres one tryptie cIeavag¢ per chain and two per molecule to appear. The region around Lys~=l,ys -~s and Lys~-Ala ~s has a low hydrophohicity and is proclieted to have a marginal a-helical content (Fig- 3). Chou-Fusmaa protein-folding i~$es predict that thi~ region should he a random cell [38,391. The immediate tormafton of peptides 55-77 and 56-77 and thalagphnse tor the other peptides st~ggest that the C-termi=lal peptides are cleaved first, after which other bonds are cleaved, The oLher pcptide bonds susceptible [o oleos,age. ¢xo:pt Lys.~ff_;lu4, al~ in regions that have a rdativcly high predicted ~helical struCture, high hyd~Jphobicity, and a high helical amphipatinc mom4mt, Also, ]ysines23, 2g. 30. 39 and 4 4 are in regions proticted to have an ex.helicalstructure in solution [38.39], The slo,,ver rate of hydtolys/s may he due to t h ~ bonds being lc&s susceptible to hydrolysis because of this s i m i a n . If the enzyme (:teared only a randomly coifed prefer.n, the appeazanc~ of the C-terminal peptid~ (which requited only one cleavage per chain) would on, or twio~ as fast as ig'ptide~ 29-39, 40-44 and 45-~4: the measured diff¢~er~ceis approx. 3.5-tiraes. Moreover, the laf, phase suggests a pt~,~fsor-product r¢latio~dtip. The results are consistent w~th the enzyme first cleaving the bonds in a razr3om-coil t~glon. This is followed by unfolding of the rest of the protein becaa~ of its low free energy of stabJliT~tion: then oth.rc bonds a~e rapidly cleaved. In comparison with f r ~ ape A-ll, ape A-II ausso¢ialed wJti~ phospbolipid has a hisher fr~o energy of =
126
one less tyro-~r~c residue I ~ chain c~ipos~T to the aqueous solmioa aaeL i-g approx. 500-1trees more rcsi~ta~l zO proteolysis. Untige ape A.[1 ia ~,]ution, the rote of aplxmCutce of peptidcs does not to[low a p t ~ r z o r prc~uct re|atiooship. The amino add analysis of apo A - ] ] ' ~s ind/~ti~guishabl¢ from d~at of ape A-IL ! ~ . rnova~ of a p~tM¢ o f residues ! - 3 ~ I d effect ~uch a ~aJ~8¢ ,,,~th~.r d~¢r/n~ the o ~ m i ; amiao ~cid aqaly~i,, The reason tho~ is not a 100~ coav~-sion or ape A-U to ape A - I V /s ~ot clear: how¢,~t, it b not an o b ~ i ~ tory step in ~ hytlro~sls d the x~.t of the prolei0. Heterogeneous eqpo A-]I asso=ation with l/pid particles or aooequivaleqooe of the ap~ A-I] e,~eins as seen by ~a~0.~ clr,~v,g.¢ or ;~po A.It in HDL 16| might at;a:oum fo~" these fimtm M, "I~e rate of dLsapF~g,"~ oJ" ~po*[[ and aPlXataa~ of several pepti&'~i ar~ virtually idonti. ca], It is difficult to di~l~mfia~ raodom hydrolysis o{ protein on the lipid surface v~rs-~ total hydrolysis 0f ape A-]I in so~utioa. Even lhottgb the ~ en~ i~ l a ~ (l~ diss0c./ation eat, from th~ I ~ i ¢ l e is nc~ known. $iat~ these exl~dagnl.~ ~ / m ~ with TOD-Fo~ ~ ~1~ o f t t y ~ n , a.~y ~ A-if wt solulio~ mmdd be hydroh-zed imm~i;~l~ly and the a p peanmce of the peptides m i d I~ id~tieal, la ar~ r.,x~, none of the tryptic cl¢,avag~ sites aye as s u s ~ i b l ~ to hydrolysis in the ] i p i d ~ a l e d protein as in solution. The ia c,r hi.gh amplti~e~'~ mameai (T~H¢ IH}. "Fh.~ve.lges ~ r the 15sire arr.lpo a~id hond~ in al~O A-l] faJl ia Ore r~,ion at sarfa¢c-associ~x~ing p e p f i ~ s 114,3~]. Th'~ higher fmqu~e~ of I~si~e ~,siduos im amphipathic hdi~al regions may be do© to ~ Lu'~ contribution of the ~sir~ residues to the ampMpathic m r . A_na.atlmrama/ah el a]. 1~21ka~ s~,eatea that the locaeuo of the lysia~ hydtophobic a'~hyka-~ gn~aps of the side-chain to in~eeact with t ~ Upki whi~ me oah,aoaium group is e~pos~ to w a ~ . t.ys~ and Lys ss ace i.n a agion d low hydroplmbi¢ity and low hel.~l pOloati~d. Ho,a~v~r+ as shovm with ~yo~etic aad trypdc pt~ptides, these re,idue, arc in l:~"plid¢~¢~g., re~dues .f~l~-'/7, that ~L~s~..iate ~ t h pb~¢4t0Lfpid ~2X].The ~ l . ~ z ~ r e l p ~ - ~ ' i ~ d ar,,so~fia,n is p'¢iu',a~y da~ to ~'~[ix (osmadon [ ~ J TFds is c o - - t a x i by a loss of conf---malone] entropy so that t ~ o ~ n t r i ~ of prolein steuctural ~ tO the fr~ ~ e r g y of lipid-prol¢in as,so6-ialioa is small. 1 ~ major contdbutio~n to the free eaeq~ of associmioa appears to bc the transfer o4' h_yd~qpbobic amitto at:~s on il~ no, poles | ~ of an ~ p : ~ i c helix from ~,.. agtz~x,~-s ,0 a ~ipoe~$k: e~zlroan'~L "i~,~ t . ~ ]~elh:al msaphipathic ~ e ~ t a n a l y ~ is a ~ g e ~ r imglich~ at ~pid-ax.~,odating don~tlS th0n methods vJ~h a.~ the Clum-Fa.~naan tales, whx:h ate based on p r o ~ foklMg a l o ~ The lysine Ia~pti~ bonds ate not ussaall~r
susceptiMc ~o ~t~/x~irt h~/~r~iyf~ i~ ape A-[I associated ~'ilh 1~hosphol~pgd. The simplest ¢xphnation is ~ s t the lack of conf0trnational freedom of the foided poly. peptide in the ,a.hclioal o0nform0fien pr¢¢cats s~soepu. hie bonds from being boand to the active site of the enz-ymc I ~ l . T'h~ pepfide bonds a r e ' buried" in that t h ~ are in f01d~ regions of the pro~c~n which are conformaIJonali~ Jna~t:~ibl© to ¢Teaveg~ "T~¢ ;,~Mli¢¢ antoanl of apoliFop~o~r, tha~ i~ c.oatotmat~OratlJ), i~aoee~6/hh: r~ k~droty~is is a d i ~ t function af ~ magnitude of t l ~ free tmergy of assodation or ~he protein with the lipid surface. Th.~ a eombinatio¢[ of sina:tu_~ pr~l~i~a methods, iac]e~li~g the helical moraem algorithm, sltoutd ellow the t i t i l l a t i o n of re~iom, o.r apolipoprcaehs u~hich ~nay be i m l x ~ a n l ie ~ote.Olytic events. Admow]edga~ats This research wa~ supported by ~r~nts from the National [esthetes of Health, HL.-262~0, Hl~.~lg"Jdr HL--~913, a_ad HL-27341 (SCOR in Artcrios¢leros~) and by a gr~r~t l r , ut The ~ - ~ l ~ &. W¢~ch F~or, dation (Q-906, ISdP). aad ~as h¢~i~,d~ from a Nation:! ~./¢nce FoundaU~n instrumemadon grant (DMB M I 3 7 5 i ) awarded to I~ylor College of ~exJ~ine. The aul]~"S wish to thank Marjori¢ Nexglham for p~p~mtion of Llxe manuscript, axM Susan KeKy fO~" providing ~ e [i~e: drawi~s.
Reteremc~ 1 Mahley. R.w_ tmm,~,ad~y,-rL, gall. $.C, 3r. stud We,gxab,n'. x.J-l. (19~j L ~pi4 Lqes-~. 127~-e~9~. 2 getPrCSL]-P.. 3~son, ~L,~ MorrJ~[t~ LD. aJ~ Goitc~. A.M., ~r, (1974~ ~ I.,;JL 18. 247-253. 3 Spanow. J.T. dmdGo,to. A.M..Jr. ItgR()pAnn. NY"~:,ad. ~:i. ~ . )&7-2It 4 W~, C-F., C~o. ~r.K,,Yc.a~ ¢..Y., g a ~ I , Y.L,. MJJae. e , Lg W~H., S ~ . J.T..GoUO. A.M..Je.and Cha~ L (lq~L%PrO¢. Natl, Acad. 5~. USA BZ -/~5-726~. lba-m~ ~R.I~a~ ~kanu,A,M. (19~) B;cChcmistry~2, 289~-2¢~. 5¢aau, A.M,, Byrmt, FLE. a~ F_ddr~/a. G. {tg~) J. Lipid I~,
7 Yt~gek. D., Ityrnr, R_I~.,Darto~s, M. ar~ Sc~m~J.&.M. (191~yJ. 9 Beadle, W~,. GilI~m, F--.ILCn'ato, ~M.. Jr. ae~1(Bantoro~j~,H. ttg~2) It/ochre. l~opltys, g¢a. Comm~:. 109,1~-12"~7. 10 Clbou, P.Y. a~ Fasman. G.D. {U;qg) Ann~_ Ray. Biatlmm. a?, 2_~]-~ H g ~ J . aad Do~]it:l~ R.F, (1~2~ ~. M~, ~L¢~- t;ST,lO.S-Z3~. t2 E/s::almr~ O, W¢~ ILM. ~d Teob;lliger. T.C. (19~|} Ni~t~ ~.99, $71-.Y~'4. 14 ~ b ~ g , O. |19~M.|Am're. Pea. ] i ~ k ~ m . 55. ~q5-623. 1~ ~ecwer, H.B.. ~,6~,$.F~ Roaim~g. ¢e,9 .k~n. K.M. (19TL~]h'~. Nml. Ar.~. ~ USA 19, 130~-t30~. 16 3¢anu, A.M. IJlgO¢~, P. =nd CI~uI~ J. 119~10)Ann. N Y Acid. ]4~. 16G-t73,
127 1"; Jc~hn. C.E.. Oshnm, J.C., Seha~l'¢r. /i.J. and BTCW~. II.R. 119:~]1
20 Men S.J.'E., Spanow. ?.T,. G]lUam. E.B,, Gogqo, A.M,. ]r, ;Ind J a e k ~ . R,L. (Jg??) ~Locbcmlutry 19, 41~-4"i_$6, 2"1 Mao, S.LT., .lacks~, R,L,, Go~lo. A,M,, Jr. sr~ Sp~reow. J.T.
~ M~s~?. J.B.. Golto. A.M.. Jr. amd Pownall. [~..J., (19791 J. 9ic~1. Che~:m. ]9. 33~q -9~61. 3~ K~i~e, E,T- ;tl~LtKe~dy, F,] (t9R3) Pt~.-, N~tL, A~nd, ~ l . USA ~r~, H37- I t4,'t. 3~: A.o~oLhttraw,ai;~h, G,M. Jones, J,L,. Brot4J]kql¢. C-,C,. Schrmd.t, C,F,. (~hLLag. B,H,. Hughe~ T,A.. ~howa, A.S an.0. Segr~sL J.F. qi9B$} 2. BitA. Chem. 260. 102~.g-1~255. 33 T~,~c,n~ll.H,J,. Knapl0, P,.D,. Ootqo, A,M,. Dr. ~nd Marcy. 3,B. (]g'83~,FEBS I.e~q. 1S9. 17 2,T ~4 K ~ s , K . E s~d Phillips. M.C. (19~)FEBS L ~ L ]?9, 2E3-26~.
7.2 Che~, T,C, ;~panow. J.'r.~ G ~ o , A,M...Tr. a n d Mrl~r.~.tl. J.D. (197~ ]~i0¢.hemistry 1L~ 1617-1622. 2] Ma:ssey, ,ES, Golm, A.M., Jr. a~l [ ~ v ~ l l , H,J, (19~C.)), Bi01. Chem. 255. -1(1197-1~17~, 24 Ma~-3,, ,,l,g,. (3o1~, A.M,, ,if-, aed Pow~a]l, H,.t', 1-1¢8118ica:hcm-
227-2~. .~6 X~Las6e~'.J.l~. a~c] I~e.,all, R I . (198fi) Mcghc,ds Et~cmol. 128. ~'4~3-,;-1fi, 37 Het~aay.s,J., Jr. (1966) J. 1~3~s.t . ~ n . 3'0, 510-515. ,~8 ~d~:ls'L,c[n,C., Kezd~,. ~.J., ,¢x'-an~A.M. and ,¢,hcn,~I.W. ,L-19"/91].
25 M m ~ 3,2~,. RoMe, M,F-, Van Winkle. W.B.. Go?to. A,M., Jr. a~xl Pow~all, HJ, H981-1 pic~homi6t~ 20, 1569-1574. ~5 I~¢ijaouA. D3".and Phillips. M.C.(19B~) 8t~'L'cmistW 23.72f~-734. 27 Lcvi~l. M, (?@?6) 3, M d . Bi~J. I K 59-107. 2~ Nog~.kL Y- and Tan(~rd.G (1971) J. Bnd. C,b~m, 2445.2211 ..2~]'& 29 R~.~. O,D.. G~4:k~wilZ, A.R.. Lesser. G.J.. Lee. ~.,H..x~:] 7~h?u,-., M.H. (~9~5) Sde~c: 229, g34~83~,
3c~ S[o(['¢1. W. (]983i i~ Pho~pholLpi~]~ ~ d Athc~6ck'f, xsi~,(Av~.~.~ro. P..Mancini. M,, g i ~ i , O, a ~ Paokl'd, g,, ndg). pp, -1l.~, 129. P,aven Pv¢-~, New ¥0~k, 4ft Pao¢. C.N,M. and Rar~elt~ ?.J. (lt~8,1) Rio~kem. I. 219. 411-417. 4] Rc~yn~h:]s.J.A. (197Gj I. Binl. Chcm. 25l. (~]2~-6105. 42 P~w~all. H.L. HJck~n. D. and Got?a. A.M.. Jr, (1981] L ]3ior, Ch~m. 254, 9g,:tg-9854.
F E ~ l~.tt. 131.3f~-363. 18 Sl~inc~nija, M.~ SasakL. N . ~l~tnhar% R,L,, Shi.~i. K, ~k~ JacksL~,l. R_L (]9~L~)]b~lfim, 13Loph?s, A¢ca ~'-13,"~q2-2~q'. 19 Kttho, M,. M a ~ w a . Y~ YQkoyama. $.. Te.jim,lL fl-. lyJ~Jkawa~ K,. Yamamoto. A_ ~acl T~ram. S. (1982) L Biochem. (Tok3.,l ';2. ~9~870,
Kinetics of tryptic hydrolysis as a probe of the structure of human p]a.¢ma apolipoprotein A-It John B, Massey, Diane LM. Hickson-Bick, Antonio M. Gotto, I1-.and Henry J. PownaH (R~v~,t 9 Ma~ 1989)
Keywards: Ap.glipopr~teit~A-It: Slrtmeu~.~'mletiu'~zelot~um~hip;"]'ryp4]chydrolssLs:(HLtmanp]a~nml A~ n model system to t m ~ alp0111mp¢oteinsirlt~tlu~d-~,~ric~,Ic,. ~, :'2q41their relatio~nsbips to Izroteolyfie event, i, the klnetl~ M tr~p4~ hytlr~ysis o f apolIpoprotetn A-JI ~apo A d D yeas i~estigated in solution anti. in ae,_,,~iati~ wlflt #hos#bo]ipid. "l'be raUs o f apt0earastce and i~ulifies o f spedfie pepddes .a,~e determined hy r * v e r ~ - p l m ~ hlgh-pe~fonmttme liquid e ] m ~ u m t o ~ , rand imago i c i d mn~]y,M~ reslu¢.edvely. F~r the kl~elle~ of h~'dpul),~ of ~,po A - [ i in solution, the e~rboxyl-termil~d i~Plides o f rasidl~es :~-7"7 and :56-7"7 apl~ flr~t, fe~lowedl hy Pel#~k"~ M' tesidhle~ 4 - 2 3 , 29-,~, 40-44 and 4S-,5,4, whid~ appe#*x'd at ncm'[y itlentieal rates~ [ ' h e ~.ioetles o f hydrolysis of apo ~ - I I m.soel~ted ,a~lb l.~dlmys[stoy]-,m'~yc'eeo-3~phosp~to¢'boline showed se'a2,m]difl'erenc-e~. Rr~l~ a lO0-I,n~lI ~ anmunt ~r ID,~in was m~edle~to oblaln a ~,imilar rate o f pt~dm-t I,mmatlno; m~cem~ a new p * q ~ appeared, elming earlier t-÷lan A - l [ ~ hm4ng tl similar llmlno aeld ¢omposl/1011;mid IIMrtLth~ mlallve hulas of aplpearmlce ot pepfides were d~'fe~_..~L ~ ~ ~'tlel'll~ Sllff~ll~illl~ ~ gontls SUL~eptil~eto Iryl~in ¢'teavage was deletmined I~- several p¢~lK'llw uJ~mridmm. The lyshie amino ~ i d bonds were folmd I~ I ~ in reglolt~ define~ by a hi~g heli¢'al mlll~ipmthic The *~luced ms~q~'bili*y to tTyp4ic by~rol~sis of sl~-II ~ i ~ * t ~ with phmsplmlip~i epp~m,s to be ~ ~o a f~ eveerl~ o f StaI~IIIzItt~OtlM I~'~ein ~oJOelmy Stlltetm-e. As. a ~ e ~ n ~ e . , the ]ysi~e amino acid I~nds are in fo~ded I ~ n s n l ~ e I~v~M~n wince tl~s'y are confornm~ons]ly ira"Ida m enxym~ie hydroid, is. 9 y xle~e o f ~ r e . p c e d i c f i o n methods, it is p4x~ihle to designate ~hieh re#ort~ of apolipopcmeins may' be important in prote&V~is,
L-nrodtmfion The prott.';,n components o[ plasma lipoproteins, termed apolipoprow.lns, distribute diffe~emly among the lipoproleins of different de.~ity classe~, gave di[fe~nt primary structures, and ~ntribute differently to the control of lipid metabohsm as activators of lipid, catabolizing taazymc:sand a.s ligands for receptor.attila, ted uptake [3_].Apolipoproteins have different metal0oliu fun~.'tloas, but ~h~r~ a common Mru~.-tutai feature; they associate with a phosphollpld/w~t~r int~f~o:, F~am analysis of tlu: primary st.tuetur¢, of apolipo,prtRei~s, a
Abbyt~L~tim~; HI~'L hbgh,d~m,~lYhl~Pe~l*ln~; ~lp~A.I, ~llcKtllpr~Oolean A-l; apo A-[l, apolipoproton A-II: UNIt'. ].Z-clLTn~nstoyt-zrnsl_',nxT~-3~,hco3~myldx~m;]IPLC,~gh-perl'mama~oellClUld~hrom~tlo~taph$:~v/s,v,",~,ighl/w¢ight:VH.S¢1i¢*1~mrp~i~hi¢moment; (SIN ltyd~patM: ( ~ ) , I~li¢.alprcbsl'dli~y: Odm-HC1.guanldi~¢ b~,d,~id¢, cm~pon6¢n~.e:J,B,Mat,~y.l.~-pa~mrn~,of MedKil~."l'h¢M~hcKIia H~lal, 6.~i~Fatwdn,M-S,A~¢OI.Houu~[011,3-X7703~,.U,$,A,
general model o f these lipid.associating domains ba.~d on tn~ concept o f an ampt~pathi~ helix was proposed [2,3], Apo-B-IO0 may bc an c'~t_'cpti,bn, hc*.:aus¢ ~;trucrural ana]ysls Lndicat*s th~1 h contains more ~-sheet st~cture than the other apolipoprotcms [41. Even with mode]s for ]ipid-proteln association, the overall slruutural organization of indivldual a p ~ i p o p ~ t~ns on the surf.ace of a lipoprotein is not known, Studies using chemical labeling, immunological meth(~ls. and proteolysts h~ve dctnonMr#.ed float epo]ipt~protr.,irrs are on the fipoprot6n sur~ra~ [.%6]. tt~hlnLiquesar~ ~.s~ulacd to diff~entiatc betwe,~t Jcg~oas that are accessible to the t~:agent ('exposed'). and regions tha r. are non-acce&~ihlo ('buried'). Correlations of these studies with the known s~que..n~¢ and p~edicted stru~.trite5 of apotlpoprot¢ins ka-~¢ h ~ n aAtb~x:~oas in such heterogeneous systems a:s native high.density lipoproteln~ which contain sevc'~a| apolipopmtcJ.ns. Mor,~2mt:r, LIt~sT~t:ili¢ Set;lu~UOC:r~gions ¢.g., antig~nlc site~, prot~lyuc cleavage sites, ctc,, of ti'w apoliproteins ~.hich am experimentally proceed have not been identified. Con~llcting reports exist concerning the extent of tryptic
1~1~7.4~.'~g/$9/.~:1.$0~: Lgg9El~ier ~:icm~*rpla0kis~h~T~B,V.ClNomedneLdD)VLb3OL3)
hydmlysi.~ of I'tDL and a p ~ H D L using sohiblc and immobilized trypsin [5]. Most studies on native H D L have indicated that a?o A-l| i~, more su~eptibie to praleolytic cleav~e than apo A-I [5,7]. Addltincally, self-as~ialion of the apeprotein ix a variable, tnvoIvemeat of pmtcolysis in "~ipol~Ol~in mclaboli~m has been demonstrated Jn the co- and post-[raoslatioaal modiGcation of the apoiipo~oteins [6÷8] and in the d e a w g e of mature apelipepre,mms on the lipopmtein panicle 17,9], To develop p~oteo~ysisas a probe of the stmctef¢ of apolipoprmeins, w e sludied the kinetics o1" tfyptic byd ~ y ~ s of apo A-II in solutiOn and comple,ted with pho~f, h01ipid~. |'he rate of appeara~e and identity of specific peptldes w e ~ delermilu~d by r~,~.rs~l.phase HPLC anti amino acM analysis, respectively. Predictive algonthms of halieal probability [10}, hydrop~thy i ] l j end helical amphipethic moment [12~14] have beam to aou|yee the ~ a n d a r y stroctare surroufid~,l~ the bonds sosceptible to tryptic cleavage. Although its runetiOns are uncertain, human apo A-It is or inlerest because it is a s.ma]t protein oonsi~fing of two id¢oti~l polypeptid¢ chains of 77 amino acids that ate linked by a single disullide bond at r e v u e (, {15}, F u r t h e ~ e . in ,dtro studies have sugi~'sted that apO A-II may regulate the activities o( [ecithio-clso]4=s~=xol acyhraasferese [16[ and hepatic |ipase [t7-191. The .~vanta~s of using apo A.]] an~ ~ a t its structure has been extensively studied w~th native and syfitl~ie peptide fragments 120-22] and that the stn~etures o[ apo A-]l/phospheliQ~d o3mplexes are well chafacteri~xl [32-251. Expednt~*~ l=,~ed~'es M~teria/s All materials and rnethc~dsfor the preparation of apo A-ll and D M P C / a p o A-II r~.ombinants ha'~e been described [23-25]. For this study, a D M P C / a p o A-l| r¢~n3binant with a lipid/psotc~n molar st~ichiometry of 7S:I was u.~d. Trypsin-TPCK (226 units/toni was from Worthington Bk~hemicals (Freehold, N J), All o t h ~ salts and solveal$ w ~ from comme¢~al ,,,~ndnrs. A sta~dar~ buffer composed or 10 mM T~is, 1(~, mM NaCt. ] m M sodium azide. I mM EDTA. 10 mM CaCI2 I'p|] 7,4) wax used throughout, Metho~ Tryptic pcpLidcs of apo A-]] w~r¢ isolated on a Spectra Phyr-~, ~O00-B high.pedmmance tlquJd thenmatog~aph with an SP-8400 variable wavelength dametar, using a C, mversed.pl~lse ,~ynchropa~h RP4 colamn ( 2 ~ x 4.1 ram). proteins and peptides were dated with a linca~ ~edient of isopropanot in ;5 mM ammonium phosphate (pH "7,3) and monitored by abs o r b a ~ at 222 rim. 1"he individual pcplides were iso. lated and i ~ t i f k ~ d by amino acid analysis,
To estimare a rate. the time for.~'~ disappearance at" the apo A . I I peak or ~tppearanee of the peptide peak wa_~ use~ a~ the ha]hime of hydrolysis (Q::). The rz/2 valt~es ~ere date.(mined by ~sual ins~liO.n of the data_ The rate of appearance of s p ~ f i c apo A-II p e p t i d ~ was de,ermined as a far~.tion of the incubation time with o3,psin. An aliquot of the sample mixtn~ containinn ~,~,sin wax collected at fegtdas iater~aL~ and quenched w~.th 6 M guanidine.HC1 a n d the~ rapidly frozen ia an ethanol/~olid CO= bath, The samples w¢]~ stot'cd frozen until analyzed by ~versed-phas¢ HPLC, Each peptid¢ was quantified, by zr~asuring the peak h~gh~_and ¢orapar~ng it to the peak height after total enzymatic hydrolysis. The int~a'ated axens of all the l~aks wese identical for each H P L C analysis, indicating the repr4~ucibility of ~ t i d e isolation on the column. To follow the ktheti¢~, the rates of c'ii~appearance o( A-II and the appearance of tryptlc peptides, residues 4-23. 29-39. 40-44, 43= 54, 55-77 a n d 56-77, were ~asure, d. Citt:alaF ~ichrGic ~ , ~ t r a were mea~tlred on a Jas, eo J-51]OA spectropolarimcter [25j and f l a o ~ r y , : e speclr~_ wine measured on a S L M 8000 spectrophotom¢4er (SLM Instrument% Urbana, IL). Thermal d~aturation of apo A . I [ / D M P C r~ombin~n~ was ]'olh~wed by Chang~ in the flaotescence intensity of sl..e ry~osir~e re'dues (etnisaion wavelength was 320 nm and excitation wa~le~g~h was 275 am) and in the molar dfipt{city me..asore~ at 222 nm ([6']~2~), The tempexattzre dependence of the speetroseopie measumm~ts was achieved by placing the thermal oouple of a Bailey Inatturnents digital thermometer (Modcl Bat ~) directly in tlre 1;uvet~ autd recording the spectral intensity vs. temperatu~. The thermos=ted sample cells w e ~ initially cooled to pr0x. 0eC by a Br;.nkman ¢i~culating. water ba~.h and the heatin8 cur~ initiating by ch~mging the thermostaq on the water bath to 100'~C which occurred OVer about 2 h. The equilibrium COnstants and standard &ee energies (.,4G°) o[ dcoaturatlon fat apo A - I I / D M P C combir~ants were calculated rron~ ~rvcs of tyrosine fluore~ence or [g]=z= vs, lamp.ratine, assuming thai the denaturation was a reversible two-s~te pr~s,~ as r,,esib©d by Rcijnoud and Phillips 1261. The secondary str~ture of a ~ A-[I was analyzed hy sa,~eral pro:|ictive algorithms which included u~t¢ helical hydrophobia momem algorithm of Eisenherg et al. q12-14]~ the hydropathy tr~hod of Kytc and Doolittle [1.l], and the helical pm~.bility as dey~ribed by, Chou aL11dFasm,an []0]. For a hydrophobicity ~al©, the .scale of L,fviu (27}, which ia based in pm't ¢~ the partitionir~g of m-nino acids between water and an organic phase E2gl, was ased, This u;ale correlates well with the buryin 8 or a Itsidue in a hydro|phobic en'.Amnm,~t j29]. A 'moving segment' of deven residues was uu~d to calculate the helical hydrophobia moment, hydmpathy ned helical probability aioutld a specific resldoe. The aver-
I~educe~P
n~.~ A-]I 5 0
I
--
-
-
-
-
~5 o
rqn~-44
,
~,
40
6o
~ ~
80
FIB. l, Tr'jpl]¢ f r e t s I7[ elpi>A - I I " ~ unatslcd by I¢~¢w-,cd-plllL~,C HPLC.~ypically, ~ 0 p j t 0 f Wocemin 1'00 p l o t I~,J/i'=l ~ontalnlng } M ~rdflt-HCI IVC~lre ~llal~e'l~ ~e!', T ~ pt~ljr~s 3~¢~c ¢1LI1¢~ " ~ l ] l a linear ~adiet~t of is
-
~ ~ 5 ~ "< ~ 10 " i~
o
..~
m,/X2
~ "F
5 • 0 0
Z
4
~
i~1~an~,
2
4
I~
~
11~. h }rig.,2. Th¢ kLn=tJc~of t.~'I~r0n hydnalygi~canapo A-El ,n ~,Mugio~wnt¢ tolt~ by [be dl:;3p~:atr#.~.J~ O1"~.~f7A-t1 {A. I~ arud Ebb"~l~l~arance
age values for 1he whole prolein were calnulated ~ the average Of the tot0.1 'TY~ovirlg sgglllcnts' p ~ ' residue for
,~f ~ e r a l IT3C~tic~pnpLide~ c~r:~ponding m r~idans ~6-T? [A. @L
the p~ot~in s e q u e n c e ,
55 .?'7 {B). 2 9 . . ~ IC.~ ~ - 2 3 (D). 4c-J,4 iE~ a ~ l ~5-_'.4 IF)- T o p~rioem Ih~" ¢~n~,~i~ml,~nl~,IIl~J]li!P~cbampl~ o4rapo-il 2~ 37~c n ~ l a i ~ -
~'qtl~l~i T h e separation of the tryptin p~ptid~s wcJ~ identified
l ) 0.r~d comparison
peptides o f a l ~
by amino
A-]I
a c i d aoaly~is ( T a b l e
to the known ~.iucnt~,
T h e di-
l O O m M NaLI. I mM EDTA. I rnM sodio.rnaz~de, l~mM CaCi~ t p H 7.45 were added to 2? ~1 of a 50:1 dilu[ic~ o] a s[ock trypsin ~olullon ¢0.8 mg/ml 3 in 0,00l ~-t HCL A[ lh¢ end g,f e:w:h ,tree ia~erv~. 6 M G d m - I I C I was ~,¢,'d It3 sl¢~Dthe rP~ction ~;~ the mLxlu~ w,~s qu;@,b' - ?1~° C I'r~'¢¢r and L I l ~ j r l ~o :~ul~mbl¢nt InmpuraLuL-¢ L~cfor¢-in3¢don ingO Iht¢ H['LC. 12unb.thin, Lion 1200 ~,l)con
TABLE I .4min,~ acid c~mpr*,~i,'.'on Amino J]¢id
r]/ re_Vphr, pe'W~.~ 4~f =,m~ A IL "
T~'D[iC ~ L ~ 4 -t:~l
~4-~g
Cy~ ~ A,p Thrb
- fl) l . t {11 - 4`25
LL8 ~1~
Glu Pro Gly Ala Val Met Ik L~u Tyr Phc Lys
,i.g {4] l j ) (15 1.3 (15
|.2 I1}
2.7 (31
2 9 - ~,9
31-. 39
J.2 qS~ I.I II}
-~.5 t3] 1,~) ( I )
t.8 IZ} 1.0 I1}
1.9 ~Z)
t.~3 (l)
1.0 (I}
40-44
I . ~ (l~
34- f',4
55-.77
f~5- 7T
0.7~il
l.I (l) - t3j
1.1 ( i } - I3]
4.2{41 1,0(ll ~, I (?]
4.2 (4} L.0{b 2,2 (.l}
2.442~ L.~it>
~ (I (25
L0(Z)
2.04:2]
2.~(2)
3.213] ~J.l~(1) ~'.0 q21 1,1 (15
3.1 q31 I,~ (l) 2-'f,I4`'~1
15.5{])
C'.9 Ii~ 1,~ 4`1) 1.7 (2) IJ~ (1) 1.6 (11
1X}(I}
0,~O)
1.8(25
1.r) 4`1}
2.0 (2~ 0.7 (l) 0l~ Ill 1.0 t l )
1.9(2)
• Fo~ amino ~ i d ar~ly~[~, 5 n n ~ l n[ tryp~ini:,~d ap.~ A - I I ~ u~'~ to L~I~U~ t k p~pii~:~ 1~" rm~cl-ph~L~C HPLC. T I ~ p~aks mca~ur~l at 220 nm ~ n c i ~ a t ~ l and s u b ~ t~ hydro])~ (24 h. 110eC) ifl ~ldc], Cv,n~uP*l¢i~[0b~. em{doyin~L 6 M HCE A~aly~is ~ pcH-orm~d un au LKB a11~11oach]5 anal~LL~r. Tla¢ valuta: rc~l'~-~lt Lhe a'a~lrag~oF th~-~ d e t ~ r m i r ~ . t i ~ : the L,'aLLI~in T J a ~ t l l ~ w aJ~ LL'I~dlRpolrL'~[~l]vullJ~. ~"C y ~ Tiffalod ~ v a ] ~ ar~ L~34LqLlanlitaL~l~ I:'APC,~PLI~C q.hc'y~ parb~.]ly dc~tr¢ly~J in lhe a~la]~2~.
I ],I
•~.ll'id,~.con~,imn B peptitJer r~$idl,~-~; 4--23i wa~; f.J.rll'Je_r i d t n t i f l ~ by r ~ c t i o n w]lh dithiothrci~l. The kinc s or hydrolysis by t~'ypsir~, 0.0O~ (w/w) tryp.
---n
,
.
t0
0 10
,C
b I
I
.
__t
D
. . .
.
.
.
.
(Ti~ 2A) ~nd 55-77 (Fi~ 2B). After p m l o o ~ d i n c u ~ zion, Chezunc~t or peplide 5~-77 db~rc&~l ~ad that or ~-77 .~d. ~r~lJneg ~y~I~i~ ~|~-~ ~?T¢ ¢se~maced by visual Lr,.~tic~n of the d~ca. The vaJ~¢ or t,/~ for apo AIII ~uas --0..~ h. while valu~ fo~ the al~raJ~ of the pcptidcs ffesidu¢~ 56-11, g/z "~ "~ h: ~d~¢ s ~5 - ~ , I { / ~ - 0 . 8 h) w©r¢ slichtly longer. The k3eetics of appearance of peptidts 4-23 (~,/~ - 3.5 h), 29-J9 (~'t/:" 3,2: h)i ~ - - ~ (t]/~ = 3-6 h)q and 45-54 (fl~ - 3,3 h) (Fig, 2C~V) exhibited a~t initial I ~ pha,~ tmtil m ~ t of the apo A-II had disappeared ~nd ptptides 55-?7 ~ d 56-'/7 had bec~ formed. The kinetics or" tt3~pti¢ hydrolysis of apo A-]I in a D M P C / a p o A-{I recombiJtan*= (75:1, ~ l / m o l ~ indiw ~ n .e~__ ~-] lq O~l~n
a
':
,"
O
'-:'='i, o
....
.~
0
k
10
20 31) ~D?'B 10 20 T|me. I~ Pill., .'I.~ : kin~Ju ud"l~'.~l~inl',),d,'rt~)'.qis¢,I'al~ A-LI "co~mbin~ w~th ~hol~d ~ follc~,'~lI~ Pal~id¢~ ! . ~ s by HPLC,T ~ ~ l t ~
s i n ' ~ g rate of .p¢"~Li4~-J'o'r,m~t.~3"l'l
w't.S the ~Tpeasan~ of an iat¢,kml.~(¢ Ip~'pd~c. apo A.II" (Fi 8, 3B), that d u ~ d tJishtty taditr than apo A-|.. Within 4~tpefimtrtl~ ©rmr ( ± ] 0 ~ ) . Uu: amino a¢itt ¢ ~ : ~ s i t i o n of this peptidc was identir,~ ~ that ol ~po .~-[I. This t~eptide may I~ve been fofme.d by hyd~i)'.~ at Lys% A third major difference w ~ in the ce~axi-,~ rates or apl~acrce o[ ~ i d ~ lro¢ alpO A-I[ a . ~ @ ~ t ~ w i ~ pho~pltolipi~, the rate of d i ~ a r a n c e apo A-ll (11/--2 -- ~.S h) and the rates 0f ~,p~rar~ce of ~ l ~ i d ~ 2 9 - 3 9 {tl/: ~ 2,5 h)+ and 4 5 - 5 4 [,l'l/z -- 4.5 It)
tl~l: clut¢~ slJEh~l~ kfum ~po A, II. A ]OI)-lrdd "niger ~ ~' ~ J n w¢~ ngCded10 ~l.~d .c~nmpmr~ble~ o[ disapl~mm~ zpo ~11 io a ~u:c~nam ~ c¢~n~-~d ~o ape A~]] free in ~ol~on.
similar to that ~r 56-77 OL/~ -- 5 It) and 5~-'77 p©pcid~ ¢ o r t e ~ o ~ l i ~ , tO ~sidacs 4-23 (t~/z - ]3 h)
TADL If |J
I ~.0
4[
AI~ ~tL/DMPC
T ~ .~e C RC'M,A~ .%-I1 ~-at ~:ik ~C.s F'i~,id I=C~
.- 19 -7 m ~9
~
22S ~
42
TABLE TII PJ'edi¢ied ~¢t't#Cd:rU:at~furc ~4~almng fhe l/yp,~iyi.~v.~ceplih?t,: y o ~ in
correlate chAxng~ ifl. [~rOtCJ~ MTucture w i t h "m~tlsnrab]e thermod~natinc qu,:nt: %:, ~;=]chas ¢qui]ibrLum col3.stant,s
ape A-It
[36] and iatnnsic surface ~ctivili¢,~ [34]As a mo~el system Io probe apotipoproteja struclurc,.functlon, the kinetics of trypllc hydrolysis of ape A - l [ in solution and a,~'~OCiated with phc~pholipid weo~ dcl(:rmined. Differences "~t¢ fecund ~n both the r,~te and order of appearan~ of differcm pcptJdes. C o t . l g a r J s o n of the twz values and relative concentra= tines of trypsin used ,show that the ape A-II is approx. 5C~-timc,s more rcgstant to protealy~s when complcxcd with phosphollpid than when free irt solution, The rate of appearance of peptidc,s in tht: proteolysts of ape A-II in solutlon follows the order ape A-[I > 55 77 ---56-77 > 4-3'3 --=29-39 ~ 40 did ~=45-54, Ape A-IJ in solution has fiL[l¢ ordcr~:d s~ruetu~ ( - 3 5 ~
gesmues Lys,_~u ( Ly~2~-Am~4
,~H= 1.16
0,56
i.as
Lys~-"~zJ as
0.95
0.9~
LTs~-SerJ~ L~-Ser ~
O.4g 0.g3
1.14 1.16
Lys~.Sef ts Lys~.Olu 47 LySa¢-Lys ~s
LOg
0.33 0.1~ 0.73 1O,2:B 0.C06 0.036 ~a.(128
Lys'-A~as~ ApoA-tI meaJXVaJue
(kgal/mol)
(I.J~ O,(~ 0,&2 0.72
{g)
{kcal/mMI
(e=)
z,l~ Z.;I z.~. 1-(]5 l.,~s 1~4
* The helJctl~.mphjf~alhicmomcnt(icK)w,%5.c.ctleuhttedbythamech~ of E~abe~g e, al,112~141,the I~top~d~? I~/'1 ) ) wus ~;ak~r~=cd f0y
mr m¢i1*~of Kyt¢and Ooatinl¢ il ll, an~ ih¢ I'ali~l INo'oabilJ~' (
u~fl~in the ~aloalat;o0~.
rates of appearance of peptides from ago A-It in solu. tion ~ different from those of ape A-1I in a DPMC/apo A-l[ (75:1, reel/mot) recombbmnt, The f ~ ~¢vgv of proton-lipid association was determined by thermal denaturation of the protein as followed by changes in tyrosine fluol~scence or ¢i~ulmdichroism. T:~ble II is a compilation of the th¢,n3aody, namic parameters for the ape A-II/pho~pholipid complex. Ape A-II in ~otation has a low fr,.x~energy oF stabilizalion, , ~ G = - - 1 kcal/mol. The free energies of the ago A.lI/phospholipid complex wo~ on the order of - 7 to - 9 kca~/mol. The e a t h a l ~ values ~etermined by thermat denaturation e l ape A - l l ~'o]lowed by changes in tyrc~ine flnoreseeagg or e[liptichy were very s/mihr to tho~c measured by batch micro, calorimetry [24.30] fez protein folding upon ]told asset'Sarian, The seoandary structure of ape A-ll was analyzed by ~vmal pl~dietivc methods, Table l i t contains the colue.g for the helical hydrophc)hie momenk the hydropathy, and the h~c;al probabilily of ao eleven-residue segment surrounding the lysinc rc.,6.duc rccognlZed as the tryps/n substrate, Disomslon The phosphalipid.a~so¢iating domains of apolipoprotelns have been d~cribod in an amphlpathlc a-helical modal 12,3] wh¢'re the polar and non-polar residues lie on ~ i l e sides of the helix and the non.pola~ fan: of helix penetrates the lipid phase. The helical amp'mpathlc moment algoritkm [t2-14,33-35]can be us=d as a quantitative refits, meat of this m o ~ l . It hag been used to ana]ye~ s4~ondary structure and to directly
a-helix) a~d a small difference in the tree el~¢zgy (~.G - - 1 kea]/mol) (Table If) between folded and deanlured states~ which is similar to ~het for the stability of a Sing]{' a-helix [37], However. ape A-ll ex]fibits certain charaetcristi~ of globular protein. For example, two tyrosine n:mdues per chain are not e s p i e d to tha aqueous solvent, as determined by solvent per~urbatJofl difference spectra (Massy, J.B. and PownaJi, H.J., unpablJshod data), The disappearance of intact ape A-It obviously would occur firs k since only one tDgtic ¢leav0ge ~ moleeo]e is required, The Formation of pept~des 55. 77 and 5 6 . 7 7 rcqinres one tryptie cIeavag¢ per chain and two per molecule to appear. The region around Lys~=l,ys -~s and Lys~-Ala ~s has a low hydrophohicity and is proclieted to have a marginal a-helical content (Fig- 3). Chou-Fusmaa protein-folding i~$es predict that thi~ region should he a random cell [38,391. The immediate tormafton of peptides 55-77 and 56-77 and thalagphnse tor the other peptides st~ggest that the C-termi=lal peptides are cleaved first, after which other bonds are cleaved, The oLher pcptide bonds susceptible [o oleos,age. ¢xo:pt Lys.~ff_;lu4, al~ in regions that have a rdativcly high predicted ~helical struCture, high hyd~Jphobicity, and a high helical amphipatinc mom4mt, Also, ]ysines23, 2g. 30. 39 and 4 4 are in regions proticted to have an ex.helicalstructure in solution [38.39], The slo,,ver rate of hydtolys/s may he due to t h ~ bonds being lc&s susceptible to hydrolysis because of this s i m i a n . If the enzyme (:teared only a randomly coifed prefer.n, the appeazanc~ of the C-terminal peptid~ (which requited only one cleavage per chain) would on, or twio~ as fast as ig'ptide~ 29-39, 40-44 and 45-~4: the measured diff¢~er~ceis approx. 3.5-tiraes. Moreover, the laf, phase suggests a pt~,~fsor-product r¢latio~dtip. The results are consistent w~th the enzyme first cleaving the bonds in a razr3om-coil t~glon. This is followed by unfolding of the rest of the protein becaa~ of its low free energy of stabJliT~tion: then oth.rc bonds a~e rapidly cleaved. In comparison with f r ~ ape A-ll, ape A-II ausso¢ialed wJti~ phospbolipid has a hisher fr~o energy of =
126
one less tyro-~r~c residue I ~ chain c~ipos~T to the aqueous solmioa aaeL i-g approx. 500-1trees more rcsi~ta~l zO proteolysis. Untige ape A.[1 ia ~,]ution, the rote of aplxmCutce of peptidcs does not to[low a p t ~ r z o r prc~uct re|atiooship. The amino add analysis of apo A - ] ] ' ~s ind/~ti~guishabl¢ from d~at of ape A-IL ! ~ . rnova~ of a p~tM¢ o f residues ! - 3 ~ I d effect ~uch a ~aJ~8¢ ,,,~th~.r d~¢r/n~ the o ~ m i ; amiao ~cid aqaly~i,, The reason tho~ is not a 100~ coav~-sion or ape A-U to ape A - I V /s ~ot clear: how¢,~t, it b not an o b ~ i ~ tory step in ~ hytlro~sls d the x~.t of the prolei0. Heterogeneous eqpo A-]I asso=ation with l/pid particles or aooequivaleqooe of the ap~ A-I] e,~eins as seen by ~a~0.~ clr,~v,g.¢ or ;~po A.It in HDL 16| might at;a:oum fo~" these fimtm M, "I~e rate of dLsapF~g,"~ oJ" ~po*[[ and aPlXataa~ of several pepti&'~i ar~ virtually idonti. ca], It is difficult to di~l~mfia~ raodom hydrolysis o{ protein on the lipid surface v~rs-~ total hydrolysis 0f ape A-]I in so~utioa. Even lhottgb the ~ en~ i~ l a ~ (l~ diss0c./ation eat, from th~ I ~ i ¢ l e is nc~ known. $iat~ these exl~dagnl.~ ~ / m ~ with TOD-Fo~ ~ ~1~ o f t t y ~ n , a.~y ~ A-if wt solulio~ mmdd be hydroh-zed imm~i;~l~ly and the a p peanmce of the peptides m i d I~ id~tieal, la ar~ r.,x~, none of the tryptic cl¢,avag~ sites aye as s u s ~ i b l ~ to hydrolysis in the ] i p i d ~ a l e d protein as in solution. The ia c,r hi.gh amplti~e~'~ mameai (T~H¢ IH}. "Fh.~ve.lges ~ r the 15sire arr.lpo a~id hond~ in al~O A-l] faJl ia Ore r~,ion at sarfa¢c-associ~x~ing p e p f i ~ s 114,3~]. Th'~ higher fmqu~e~ of I~si~e ~,siduos im amphipathic hdi~al regions may be do© to ~ Lu'~ contribution of the ~sir~ residues to the ampMpathic m r . A_na.atlmrama/ah el a]. 1~21ka~ s~,eatea that the locaeuo of the lysia~ hydtophobic a'~hyka-~ gn~aps of the side-chain to in~eeact with t ~ Upki whi~ me oah,aoaium group is e~pos~ to w a ~ . t.ys~ and Lys ss ace i.n a agion d low hydroplmbi¢ity and low hel.~l pOloati~d. Ho,a~v~r+ as shovm with ~yo~etic aad trypdc pt~ptides, these re,idue, arc in l:~"plid¢~¢~g., re~dues .f~l~-'/7, that ~L~s~..iate ~ t h pb~¢4t0Lfpid ~2X].The ~ l . ~ z ~ r e l p ~ - ~ ' i ~ d ar,,so~fia,n is p'¢iu',a~y da~ to ~'~[ix (osmadon [ ~ J TFds is c o - - t a x i by a loss of conf---malone] entropy so that t ~ o ~ n t r i ~ of prolein steuctural ~ tO the fr~ ~ e r g y of lipid-prol¢in as,so6-ialioa is small. 1 ~ major contdbutio~n to the free eaeq~ of associmioa appears to bc the transfer o4' h_yd~qpbobic amitto at:~s on il~ no, poles | ~ of an ~ p : ~ i c helix from ~,.. agtz~x,~-s ,0 a ~ipoe~$k: e~zlroan'~L "i~,~ t . ~ ]~elh:al msaphipathic ~ e ~ t a n a l y ~ is a ~ g e ~ r imglich~ at ~pid-ax.~,odating don~tlS th0n methods vJ~h a.~ the Clum-Fa.~naan tales, whx:h ate based on p r o ~ foklMg a l o ~ The lysine Ia~pti~ bonds ate not ussaall~r
susceptiMc ~o ~t~/x~irt h~/~r~iyf~ i~ ape A-[I associated ~'ilh 1~hosphol~pgd. The simplest ¢xphnation is ~ s t the lack of conf0trnational freedom of the foided poly. peptide in the ,a.hclioal o0nform0fien pr¢¢cats s~soepu. hie bonds from being boand to the active site of the enz-ymc I ~ l . T'h~ pepfide bonds a r e ' buried" in that t h ~ are in f01d~ regions of the pro~c~n which are conformaIJonali~ Jna~t:~ibl© to ¢Teaveg~ "T~¢ ;,~Mli¢¢ antoanl of apoliFop~o~r, tha~ i~ c.oatotmat~OratlJ), i~aoee~6/hh: r~ k~droty~is is a d i ~ t function af ~ magnitude of t l ~ free tmergy of assodation or ~he protein with the lipid surface. Th.~ a eombinatio¢[ of sina:tu_~ pr~l~i~a methods, iac]e~li~g the helical moraem algorithm, sltoutd ellow the t i t i l l a t i o n of re~iom, o.r apolipoprcaehs u~hich ~nay be i m l x ~ a n l ie ~ote.Olytic events. Admow]edga~ats This research wa~ supported by ~r~nts from the National [esthetes of Health, HL.-262~0, Hl~.~lg"Jdr HL--~913, a_ad HL-27341 (SCOR in Artcrios¢leros~) and by a gr~r~t l r , ut The ~ - ~ l ~ &. W¢~ch F~or, dation (Q-906, ISdP). aad ~as h¢~i~,d~ from a Nation:! ~./¢nce FoundaU~n instrumemadon grant (DMB M I 3 7 5 i ) awarded to I~ylor College of ~exJ~ine. The aul]~"S wish to thank Marjori¢ Nexglham for p~p~mtion of Llxe manuscript, axM Susan KeKy fO~" providing ~ e [i~e: drawi~s.
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