Cross-bridge movement and the conformational state of the myosin hinge in skeletal muscle

Cross-bridge Movement and the Conformational of the Myosin Hinge in Skeletal Muscle HITOSHI

I_~ENO ANI) WILLIAM

State

P. HARKINGTO?;

IZ’e have modified and extended our earlier procedure for cross-linking myosin segments in t,he thick filaments of muscle so as to: (1) follow the time-course of erosslinking the subfragmentand light meromyosin regions as a function of pH ; and (2) eliminate the contribution of intramolecular cross-linking in the analysis of the sodium dodecyl sulfate/polyacrylamide gels. From the kinetics of cross-hnking t,he S-2+ and LMM regions of myosln in glycerinat’ed rigor myofibrils of rabbit \ve find that the normalized rate (k,,,‘k,,m,) of cross-linking falls over a narrow range of pH (7.4 to 8.4) with a sigmoidal profile closely similar to that observed earlier for the pH dependence of cross-linking of the subfragment-l subunits. This result is contrary to our earlier conclusion (Sutoh rt al., 197% ; Chiao 8: Harrington, 1979) that. a major fraction of the S-2 link is immobilized on the surface under these conditions. and suggests that this segment, like the S-l subunit, is released and swings away from the thick filament surface when the pH is raised. This release of S-2 is highly co-operative and appears to be accompanied by a eonformational transition of the polypept)ide chains within the light meromyosinheavy meromyosin hinge to a more open structure, as shown by an increased susceptrbilitg to chymot,ryptic proteolysis. Treatment of the r-o-operative release process as a two-state equilibrium reveals that’ a change in charge equivalent to release of only about two protons occurs when the cross-bridge is detached from t,he surface of the thick filament.

1. Introduction Our rrernt experiments (Chiao & Harrington, 1979) on glycerinated skeletal muscle fibers in rigor showed that the myosin heads could be cross-linked to the backbone of’ the thick filament while they are still attached to the thin filament. We demonstrated further that the heads could be made to move out from the thick filarnrnt~ by a small change in pH from 7.4 to 8-O. This behavior suggests that relatively small changes in the local ionic environment can release the cross-bridge from the surface of the thick filament. The possibility that such a release could lead t,o the generation of force; through an n-helix-random coil transition in the subfragmentregion of myosin during a cross-bridge cycle, has been considered in t Ablm~viations used: S-2. subfragment~2; LMM. light mrromyosin; HMM, heavy meromyosin: S-I. ~ubfragrnrv~t-1 : SDS. sodium dodecyl sulfate: TIMS. dimethyl suberimidate: k!j ,. the ratio of the 2 crosslinking ratta constnnts (k, Jkrod); k!j,. t,he ratio of the 2 wowlinking rate constants (k, P/k,A,,.\lM). Cl9 (H)“L’~%X:~fi/‘81;~(~1~~-~~ $W.oo/()

0 1981 Academiv Press Inc. (London)

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earlier papers (Harrington, 1971.1979: Tsong rf ~1.. 1979) and is supported by thermal melting experiments on the S-2+ fragments isolated from rabbit skeletal myosin. At low t’emperature (- 0°C”) the long S-2 (Weeds & Pope, 1977) exhibits a high degree of l-helical coiled-coil structure. but in solution under physiological conditions (37”(‘), about 25% of t,his conformational pattern is melted to a random roil (Sutoh rt al., 197%). Most of the melting in this structure appears to occur in t,he LMPlGHMM “hinge“ region (Harrington d CL/..unpublished data), which makes up about one-third of the length of t,he long S-2. Hence. if this segment was stabilized in the ,x-helical state when the head is close to the filament backbone. but becomes destabilized following at,tachment of the head to the thin filament during a cross-bridge cycle. t,he consequent partial rnelt’ing of the S-2 segment would produce enough shortening force to account for the tension developed in active muscle (Harrington. 1979). In our earlier studies (Sutoh ct nl.. 197%: (‘hiao K- Harrington. 1979) a rna,jor fraction of the S-2 link appeared to be immobilized on the thick filament, surface. even when the S-l subunits were released and swung away from the surface. This conclusion was based on kinetic evidence showing that the rate of cross-linking the HMM segment of myosin to the thick filament backbone, with dimethyl suberimidate. was about the same as that of LMM at pH 8.3, where the myosin heads are displaced away from the surface. In this paper we reinvestigate the question of the release of the cross-bridge by directly following the time-course of cross-linking the S-2 segment to the backbone as a function of pH. We have also probed for conformational transitions in the HMM-LMM hinge region, based on the susceptibility of this region to proteolytic cleavage. which could be correlated with the release of the cross-bridge. Our studies suggest that the S-2 segment is released and swings away from the thick filament, on elevating the pH over the same narrow range (pH 74 to 84). where the myosin heads are detached from the thick filament surface as shown by the earlier studies. The release of the S-2 link appears to be a highly co-operative process and is accompanied bg a conformational transition to a more open. proteolytically sensitive structure within the LMMHMM hinge region of the S-2 link. Assuming a t,wo-state transition. we estimate that about t,wo protons are dissociated when the cross-bridge is detached from the thick filament surface.

2. Materials and Methods (a)

Preparation

of glycrrinat~d

myojihrils

Ra’rjhit psoas glycrrinat,ed muscle was prepared following the procedure of Rome (1967) muscle was teased and and stored at -20°C for 3 to 5 months before use. The glyoerinated cut into sections about 2 mm in diameter and 5 mm in length. Fiber bundles were homogenized in a Sorvall Micro-Omnimixcr for 4.5 s at 60 V in 80 mM-NaCl. 40 mMimidazole.HU (pH 7.0) at 5°C. The myofibrils thus prepared were collected by centrifugation at about, 2000 revs/min for 3 min in a clinical, desk-top centrifuge. This procedure, including homogenization, was repeated 3 times. Samples of myofibrillar mass were washed 3 times ait,h 10 vol. buffer solution appropriate for each experiment. The approximate concentration of prot,ein was determined by dissolving the myofibrils in 5”& (w/r) sodium dodecyl sulfate and measuring the absorbance at 280 nm, assuming E&$,., 1 7 t See footnote

to 1’. 619.

(‘Ross--KKII)(:E:

MOVEMES’I’

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THE

MYOSIN

(ill

HINGE

(Sutoh & Harrington. 1977). Dissolved myofibrils were passed through filter papet (IVhatman 40) Iwfore determining t,he protein concentration. to remove undissolved material The sarcomew length of the rigor myotihrils (2.5r)@3 pm) used in this study was determined wit,h a S&on phase-con&-ast, microscope. Approximately 85?,, of myosin heads are \mund to a&in at this sarcomere length (Cooke & Franks, 1980: Lovell & Harringtorr. l!%w). (I))

f’rrparatiotf

Myosin HSISI. S-1, LMM, SlltotI /,I rd. (I97Xh) (v)

(‘rawlinkitcg

of myosin

srpuuts

rod and low molecular

of acti,/--sctl,fmgmr,tt-l

from

weight

complex

pl1r~fird

myosiu

S-2 were prepared

(ado-R-1)

according

to

in myqfibrils

III 0rtic.r t.o ir1vrstigat.e the binding capacit? of S-l t,o actin filaments before and after reaction with DMS. myofibrils (4 mg protem/ml) were digested with -\-chymotrypsin (I : 25, \v/\v) in 40 mwimidazole~ HCI (pH i.(I), 80 mrv-Ka(“l. 5 mM-EDTA at 20”(’ for 20 min. Ihgwtiorr \vas terminated by addit,ion of phenylmethylsulfonyl fluoride (final concn. 2 rnlf). Digested myofihrils were mixed with D1IS (final concn, 1 mg/ml) and the cross-linking rcbaction \vas carried out at 2O’Y’ for up t,o 30 min. (boss-linking was quenched by addition of 0.1 vol. OCZwethanolamine. HC’I (pH 7.0). Samples at various stages of cross-linking were centrifuged (IOO.oWg for 60min) and the supernatant and pellet fractions were examined I~? (,lrct,roF)hort~sis on SDS-containing gels.

>lyotiI)rils at 40 mg protein/ml in either 40 rnnf-imidazole HCI (pH 7.0 t)o 7.4) or 40 mw tl,iethanolamint,. HC’I (pH 7.4 to 8%) were cross-linked with DMS for LIP to 3 h at various pH values het\+wrr 7.0 and 85. At each pH examined. DMS was dissolved in the appropriate I)uffrr. and the pH was adjusted with 2 M-NaOH. One ml of DMS solution q-as then added to 9 ml of myofiln-il suspension. Since thr rate of amidination of Iysine side-chains has a positive pH dependence around pH 7.0 to 8.5 (Hunter 8r Ludwig. 1962), the final concentration of cross-linker was gradually reduced from 3.2 mg/ml (12 mM) at pH 7.0 to @B mg/ml (2 mM) at pH 8% to maintain about the same rate of cwss-linking. (‘ross-linking was carried out at 5Y’. unless otherwise statcxd. ~+it,h wtlstant st,irring. The cross-linking reaction was terminated at various times by th(> addition of 9 ml of 0.2 .wethanolamine. 20 mw-imidazole (adjusted to pH 7-O with HC’I) to a I ml sample of reaction mixture. The mixt)ure was left for 30 min and the myofibrils collect,ed in a calinic*al centrifuge. The precipitattb ( -01 ml) was resuspendrd in 6 ml of Cl wNaCI, I mwimidazole H(‘I (pH 7.0) and, aft,er wntrifugation. resuspended in 1 ml of 0.1 wNa(‘l, I mwimid~zok~. H(‘1 (pH 7.0) and stored at, 5”(“.

IX%1 \vas prepared from myofibrils at various stages of cross-linking as follows: myotibrils (3 to 4 mg protcGl/ml) in WI wKa(‘l. I mwimidazole. HCI (pH 7.0) wrre mixed with an equal WI umr of 80 rn>l-triethanolamine. HO (pH Sri), I .2 wh’ac’l, 2 mn-Call,, and Ic.hymotrypsin was added to give a final enzyme to substrate ratio of 1 : 50 (H./W). The high concentration of salt in this medium (0.6 31) results in rapid proteolytic cleavage of myosin into HMhl and LMM fragments. When the myofibrils are digested-in the lowsalt medium used for cross-linking, very little HMM or LMM is formed (Weeds 8r Pope, 1977). The digestion at pH 8.5 was allowed to proceed for 30 min to produce HMM and LMM. To preparc short S-2, a sample of t,his solution was allowed to cont,inue digestion t)g chhymotrypsin for 15 min following addition of 0.1 vol. 100 mwEDTA (pH 7.0). The junction

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Iwtuwn S-l and S-2 is cleaved at this step, wleasing thv long S-4 tail fragment, from HhlM (LVcrds K: Pope. 1977). Trypsin, dissolved in unbuffered wat,er. was then added to this solution (I : 25. \v/w) t,o allow furthrr digestion of long S-2 to short, S-2 fragments. Tryptic digestion was allowd to proceed for IO min. I-ttdtlr t,hrsr conditions LMJT is completely cwttwrt~ed to thts IOM molecular weight LMM fragment, ,411 digestions ~vt‘rv varrird out, at PO‘V and proteolysis was terminated Ivy adding O%G vol. ~)hrr~~lmeth~lsnlfonyl fuoridr (F,, in 2-propanol). Spwial cart’ was taken to identify thta LJIhl, H,1IJI and short S-2 fragments lm~patwi from tnyofibrils. l~‘our ttwthods w.ere used for the idvntilicat~ion. (I ) Elect.rophutwis of myosin segments on SDS-containing gels was performed awording to the met,hod of Laemmli (1970). Elwtrophorc~tiv mof)ilities were compared to stattdard marker proteins. which wtw various aut,hentic mposin segments prepared from purified mvosin using \~t,Il-c,stahlishrd methods. (2) The alcohol-denaturat,iort method of Szrnt~(:yiirgyt vt nl. (fQ60) was used to drrtatuw thv globular prot,rins of the myofibrils irretersil)l,yl Mynfiltril suqwnsions Were mixed with a 3 fold volume\ of al)solut,c c%hanol, and thv prwtptt,atr was collwtrd using a c*linical centrifuge,. The pellet was suspended in 0% nt-NaC”l, 20 mwimidazolr HC’I (pH 7.0) to giw a final prot,ein concentration of 1 to 2 mg/ml. Dtmaturt,d protein was removed t)y wntrifugation. and the supernatant fract,iott cont,aining segments of the rod portion of myosin examined t)?t~lrc~trophorrsis on SDS-containing gels. (3) Th e solnl~ility of thv various tnyosin srgmrnts was t&rd in IOU ionic st,rrngth t)uffers. Myofihrillar protein digested with I-chymotrypsitt and trypsirr \vas dialyzed against I mM-Mg(‘l,. I mwsodium pprophosphat,e. 10 rnw imidazole. H(‘1 (pH 7.0) at 5°C’ overnight. No further prot,eolytic digestion was olwwvd during ckxt,txnsirck dialysis. Thv clia1ysat.r was centrifuged at 5°C’ (100.000 g for QO min) and the supt~rnatant. fraction r~xaminrd I)y t,lec!t,l,ol)horc,sis 01, SDS-containing gels. Bands c~orrrsponding to the HM.lJ. S-l and S-2 \vt’rtl otwrrrd in t)hv supwttatant, fraction. Thrl t)atrds corresponding t,o LMM. Hhlnl and short S-2 prepared from myofilwils were found to iw indistinguishablt from the \~rlI~charactcrizr,d myosin scgmcnts prepared from purified myosin. (1) Short S-2 was distinguished from low mofewlar \vvightj LMM by its ability t,o fortn a disulfide-bridged molrculr. Low molwular \veight LMM fragments were prepared t)y digestion of isolated LMM with trypsin followed 1,~ oxidation of SH groups in 0% wNa(‘f. as dt~scrittrd itt thts next section. and showed no formatiotl of disulfide-bridged spwies \vhrtt examined by electrophorrsis on Sl)S/polvactl;lamiclr gels. In contrast. both long LM&l and short S-P fragmtv1t.s were ablr to form disltlfiti~,-l)ridg~,~l dimers on thv gels (SW Fig. I ). To see if the protrotytic digest,ion of myosin molewfrs is affected fly wowlinks. myofibrils \v(‘re cross-linked with the cleavable I)ifunc~tiottal rtaagrnt dimethyt 3,3’-dithiobispropioniI)y digest,ion with \\ midate, in high (I = 0%) and low (I = @05) salt at pH 7 fblloaed chymotrypsin at J)H 8-5 (r = OS) t.o clravc~ t,hc H&J&l ~LMM hinge region. Whrn tngofilwils \vvw cross-linked in high salt. the rate of digestion after c&rowlinking was comparable to that in t,hr atwnce of cross-links. Since itttramolec~utar cross-links were dominant in this case the! do trot swm to affect the digestion. When myofihrils were cross-linked in low salt, which favors intermolecular cross-linking. more t,hatt !#5(+o of t,hr myosin chains were cleaved (I = 0%) at thr st,age N-hew -!W, of LMhl was intt~rmolwularly cross-linked. (f) Oxidrrtiot/

of thr

SH

groccps

of thr sctt~frtrgmr~ct-2

(II/~

light

n~rromyosin

frcrymr~rts

specific WT’r oxidized the SH groups in S-2. LMM art4 rod itt order to introduce intramolecular S-S bonds into these fragmrnts of myositt. The procedure of Stewart (1975) \vas modified for use in this study. We used this twhniqw to dist,ittguish intramolecular cross-links from intermolecular cross-links formed in the reaction with DMS. During t’he course of this study WY compared the cross-linking of myofihrils with DMS in physiological (I = WI ) and at high (I = 0.6) ionic strength conditions. Thr rate of cross-linking m?osin heavy chaitts with DIMS was found t,o occur as fast in the filament system at low tonic strength as in the dispersed myosin at high ionic+ strength. This suggests that intramolecular cross-links may be formed in thr myofihril systrm at a rate comparable to the formation of intrrmolecular cww-links. It was thrrefow newwary to drvisr procedutw for detecting only

(‘ROSS-I~Rll)(:E

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THE

IvlYOSIS

HIS(:E

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illtPl.rrloltl(‘Illar vross-links be~wtwt neighboring tnolecules of the thick filament, core in order tq) follow t.hr outward movement of thv S-2 link. Two methods %\ere used t,o monitor itttrrmol~~c~rrlar cross-linked spwies. In otter method we used gel r~lwtro~~horwis under, notes ctc~naturing conditions in the presence of pyrophosphatr to det,ect’ native monomeric %chaitr species. We also employed SDS/polyacrrlamidr gel electrophoresis to detect disulfid(B Ilridged spwiw in \vhich intramolecular s--S bonds were introduced, Both methods gave similar results Ijut the SDS/polvacrylantidt, gel electrophoresis method was easier to perform atd sho\ved highrt, reproducibility. We therefore used this procedure rxrlusively to follon the time-course of cross-linking the S-2 and LMM segments of myosin in the rigor m~ofibrils ()xidatiotl of SH groups was performed as follows. After digestion of the myofibrtls itt tbv Jtigh-salt. tnt~dium (O-6 wNaC1, 1 mzv-(“a(‘l,, 40 mm-trirt.hartolamittf,. pH H3), the enzymes 15C~I‘Virtac~tivatcd u ith lthrtt~lmethylsulfott~l Huoridr and t.he susprttsiott mixed \vtth a solution vortt~aitlitrp 0-I wVu(‘l, and 02 wTr& (pH adjusted t,o 7) to give a final free conctt of IO mw(‘u2’ M:cx t.outinely used a 10 mitt oxidat,iott t)imr in this stttdy. The oxidattott tvactiott was t~wminated by adding 0.1 vol. 0.1 wiodoawtatr. 0.1 ~1-EDT.L\. The same l)row~drtre \vas used for oxidation of purified myosin schgtnents.

\Y(t employed slab gvls 11 cm x 14 cm x O-l.5 cm (each sample loading \vell was 0% pm in \\idth) and a Tris-glycitte system according to t.ha m&hod of Laemmli (1970) ur thrl lthrtsphatt~ buffer system according to \Vrbrr & Osborn (1969). Protein samples were dvnatureti I)y adding an approximat,eiy equal volume of SDS solution ( 10?10 (w/v) SI)S 02 >t-Tris. HC”I (pH 6.8). with or without 04)loo (w/v) bromphenol blue, :300,, (v/v) glywrol. I”,, (v/v) %-tnercaptoethano1) to give a final cottcn of I to 2 mg/ml. Samples were heated in boiling water for 3 to 5 min immediately after addition of t,he SDK solution. St,aittinp and (lextaining procedures \vere as described by Laemmli ( 1970). (‘oomassie brilliant blue K250 was uwd to stain protein bands. The absorbance of the protein bands at 550 nm was clrt.nminrd with a (illford gel-scanner and thv areas under densitometer t,racings of protritt Iw~tix \I:w( measured by wvighing the peaks excised from the chart paper. The myosin and wtin bands were fitutrd to obey Beer’s law below 30 pg total loading mass of glycerinatrd myotibrillat~ prot)etn (it4 loadings wcw adjusted to ensure that, each band aft,rt (,lec,trol)hor(,sis cottbained less than 20 pg of mat,erial when measurements of absorbance wert’ t.arric*tl out

Slab gels contitinrd 0.1 #-sodium pyrophosphate, I mM-EDTX (pH 84), PO (w/v) aw~ylamitfc~ and 013(& (w/v) meth~IPne~bisacr~Iam~~~~. The &c&rode chambers were filled with a solution consisting of 0.1 M-sodium pyrophosphate. 1 mM-EDTA (pH 8.0). This solvent system ensured that various myositt segments migrated in the gel without association betwwn monomeric species. Before applicat)ion of the protein solut.ions. residual ammonium pt~rsulfate was removed from the gels by applying a potential of 20 V for I h l’rot~c~itt solutions \vere dialyzed against 0.1 M-sodium pyrophosphatc. 5 mwEDTA (pH 8.0). .5~~oglycerol, before loading on the gels. Electrophore~is at 5°C \vas performed for 18 h at 30 \‘. Othvt, cwndit ions were similar to those drwribed in section (g), above. ( i)

Kirteticn

c~f ~ro.ss-lit)

kltc
nzyosir,

wgmcn

ts

(:Iywrittated myofibrils wew cross-linked with DMS at various pH values. as described it1 section (d). abow. The titne-courses of cross-linking various myosin segments were obtained l)y digesting samples of the cross-linked myofibrils as described in section (e), above. The kinrt,ics of cross-linking each myosin segment were determined frotn densitometry of each segment, band observed in the gels (Sutoh & Harrington, 1977: Sutoh rt al., 1978u: (‘hiao 6 Harrington. 1979) The kinetics of vross-linking LMM and short S-2 were obtairwd

624

H. I’ENO

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individually by digesting cross-linked myofbrils with enzymes at pH 85. as described in section (e). above. followed I)?; oxidation of the SH groups in these segments, as described in section (f), above. M?;ofibrils containing HMM and LMM fragments were oxidized to quantitate disulfide-hrtdged LMM. and those containing short S-2 and low molecular weight LMM fragment’s were oxidized t,o quantitate disulfide-bridged short S-2. The oxidation reaction allowed random formation of intermolecular S-S bonds to occur between the globular pro’oteins of the myofibrils, which wre t.hett trapped on t,op of the gels. Standard prot,t>ins tvere trot, used to calibrate the band densities itt these studies. since wr found that the error in loading protein mass was 5 to IO”,,. whirh was within the experimental error of obtaining rates of cross-linking (see Fig. 5). The ratio of the rates of czross-linking subfragmentand rod, k,: ,. was det,ermined as described 1)~ (‘hiao & Harrington (1979).

Myofil)rils \verc suspended in 40 mat-imidazolts H(‘l (pH 7.0 to 7.4), 30 mw?ia(‘l. 0.1 mw &IgC’l, or in 40 mM-t,rietttanolamine’ HVI (pH 7.4 t.o 8.5), IO mwSaCI, 0.1 mu-MgCl, at a myofil)rillar protein concentration of I.0 mg/ml v(‘hymotryptic digestion of myofihrils was carried out at an enzyme to substrate weight rat.io of I : IO at 5°C’ and I : 30 at 2OY’. since we assumcld that the enzymatic activity at 2OY’ would Iw about, 3 times as large as that at 5,“(‘. Digestion was stopped at vartous stages of thr waction by adding 0.1 vol. ph~~~i~lrneth~lsulfor~~l fluoride (P, in 2-propanol) to samples of the reacting system. The digested tnaterial was then examined hy elect~rophoresis on SDS-containing gels. The absorbance of the myosin heavy chain and LMM bands was recorded as described in w&ion (g). above. Thv values were normalized by dividing ly the total absorbance of the myosin bands. which migrated slower than a&in on SDS-containing gels. Assignment of the LMM band was carried out as dcsc*Clwtl in srctiott (e). abow. AaIl the clearage reactions showed single expom~ntial behavior and ra.te constants were obtained from linear log absorhanc~e WTSIIStime plots. When myofibrils were digested at 5°C”. the rate constant of cleavage of t,he myosin heavy chain (yH,-) and the rate constant of formation of LMM (yLMM) were the same within experimental error at each pH value. In this case, the rate of cleavage at, t,he myosin hinge region was expressed ty the cleavage rate of the myosin heavy chain. Ott the other hand. when the myofibrils \vere dtgestsd at 20°C. a broad spectrum of products was seen around t,hc mposin heavy chain and HMJI hands in the pH range 7.0 to 5.5. and N t’ t,herefore used thv rat<, of formation of LMM t.o follow cleavage at t,ht; hinge in t,his ray+*. HMM attd IAM \svre the, major digestion prodtwt.s at. 20°C’ Iwtwwn pH i-6 and pH 86. and Mr measured yHC. \vhicth was very c~loset,o yLMM itt t)his wgion, to follow cleavage at tht, HJfiV I,MM hinge. The intrinsic rate of peptide bond cleavage I)y c*hymotrypsin \-arias by about) 409, over thv pH range 7.0 t,o 8% (HPSS. 1971 ). and t)he digrstiotr rat,e con&ants obtained in t,he present study have therefort) Iteen csorrwted for thv pH dependence of the enzyme (see Fig. 9). (lorrcctions of t,he rat,es \vrre tnade relative t,o the c*leavagc rate at pH X.0 (opt,imum pH of ttir enzyme).

from bVorthingtot1 Rioc~hemical. All Trypsitt and y-chymotrppsin \vt‘re pur&ased chemicals used in this st.udy were reagent, grade. Imidazole. HC’I buffers were prepared by addition of HC’I to a solution of the free base. Trirthanolamitte~ HC’I buffers were prepared by addit,iott of Ka0H to a solution of triet,hanolamine hydrochloride. 3. Results (a) (:Ptlrtvl I II this study we have investigated out\vard movement of the HMM myosin molecwles from the thick filaments of glycerinated myofibrils

segments of in rigor by

(‘t:OSS-Ht
MO\‘EhlES’I’

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MUOSIS

HISGE

ti2*5

monit,oring the kinetics of cross-linking the S-2 region to the thick filament backbone \vit.h dimethgl suberimidate. As in our earlier work on the radial disposit,ion of myosin heads, we expected the rate of the cross-linking reaction t’o be a sensitive indicator of the spatial proximity of this segment to the thick filament surface. Following the general procedure of the earlier studies, we quenched the reaction at various stages of cross-linking. cleaved the mgosin molecule into its subfragments with an appropriate protease and determined the time-course of c*ross-liilking each subfragment region to the thick filament surface by measuring t.hv decay in absorbance of t,he corresponding band following electrophoresis of the digest, on SI)S/f~olvacr~lamide gels. In the case of the LMM and S-2 regions of m>,osin. it is ntwssarg to establish clearly that the time-dependent loss of densit! on the gels results from cross-linking to neighboring molecules within the thick filament vow. Otherwise, the relative mobility of these structural regions of the thick filament cannot be d&ermined unambiguously. In earlier studies of synthetic t’hick filaments (Sutoh ut al.. 197Xn) and glycwinated psoas muscle fibers ((‘hiao K: Harrington. 197!)). the rate of crosslinking the HMM segment. of myosin to the t,hick fila.ment, backbone with dimethyl subrrimidate was found to be about, the same as that of LMM at pH X.3, where t)hc myosin heads are displaced away from the surface. Since the S-2 region comprises about XP,, of t,he rod, these results suggested that a significant portion of S-2 is vlosr to the filament surface under these conditions. However, the rates of crosslinking LMSl and HINLM were not determined at lower pH values near neutrality in thtw experiments. since protcolytic cleavage of myosin filaments at the LMPolHMSI junction is extremely slow at, pH 7 (Weeds & I’ope. 1977). Additionally. ~~~~t~twlysis under these condit,ions yields a digest showing complex band patterns on Sl)S-containing gels. making quantitat,ive measur>ments on well-defined frapmrnt~s impossible. To circumvcwt these problems we compared, in the earl? stages of this work. the rates of cross-linking HMM and LMM in native thick tilarnent,s over this pH range as follows: myofibrils were cross-linked at low ionic. strength and low temperature (I = OG5, 5°C’) then digestsed with ,I-chymotrypsin at high ionic strength and high temperature (I = O+i, 20°C’) to form well-defined HMM itn(l LMM suhfragments. We found that the relative cross-linking rate of HMM t,o that of LM~~l. kHMM/kLMM, decreased about 2P. ,,,, on increasing the pH of crosslinking from i to X.5 (k,,,/k,,, was 1% + 0.1 and 1.2 +O.l, resp&ively), suggesting outward morernvnt of HMM from the thick filament surface. But from these da,ta alon<’ we \vprtl unable t,o determine whether this effect results from a decrease in t)hr rate cwnstant ofvross-linking the myosin heads to the thick filament surface, k, , or from both k, , and k,~, relative to LBlhl with increasing pH. .I significant slumber of intramolecular cross-links within the HMM and LIV>l segments may also have been formed during cross-linking of the myofibrils. Sinw \vt’ hxw drtt~rtninrd the relative absorbance of the myosin subfragment Iwlypeptide chains on SIX-containing gels. any intramolecular-linked chains will cwntributr to the time-dependent decay of the corresponding monomer chain. We hare therefore modified the cross-linking procedure employed earlier in order to follo\v the rate of cross-linking S-2 directly, and hence t)o determine if this segment of myosill is released from the thick filament, surface on elevating the pH,

ti”ti

H.

I’lCSO

ASI)

\\‘.

F.

H.AliI~IN(:‘I’OS

Rabbit, psoas myotibrils prepared from glywrinat,cd muscle \vere c+ross-linked \vith I)kIS in dilute salt. solutions at diff6wnt pH valws owr the rqy ‘7 to 8.5 (5 ‘( ‘). ‘-it various stages of the waction. samples wcrc~ wlnovcyl from thv rtywt,jng system and \VPI’P tjhen digested with chymotrypsin in the presency of l m1l-C ‘a2 + ill 0% JI-N~( ‘I at’ l)H 8.5 (20°C‘) aRet. clurnching t.hr cross-linkirlg reactiorj. [‘ndrr theses sol\.rt~t. conditiws only t.he I,iM~l HN>l jrlnction of the> myosin mcmomrr is rapid!> &%\Nl. l’\vo additional strljs wew used to follow t,hr cross-linking rate of t,he S-2 wgment~ of myosin. (I ) Addition of EDTX allows cshymotr>,psin in the HSIM-LMM mixturr t,o clt~aw HMM a.t thr> S-I /S-l junct,ion (W;rrds A I’op~~. 1Wi). (2) Furt,her digestion of thv wsulting long S-2 with trypin removes t,ht, IA&IGHMJI hingcb. yit4ding the short S-Z (Srltoh rt trl.. 19786). WV have followed tht timr-course of cross-linking short S-2 rathrr t,han long S-2 hecause this fragment is well-resolved from IAIM on tIlti SI)S-~ont.aitlillg grts and also hwausr short S-2 is mow stable than long S-2 during taxt,rnsivv pivttwlysis. 1‘0 wsure that \ve wert~ monitoring i~ltrrmolrcular woss-linking rather than i~~t,t~atrlolr~llli~r cross-linking Iwt,uc~ct~ thtt indi\-idual polypeptide c4laitis of a trtolwulc. we oxidized the I,MM and short S-d fragment,s in a high-salt (0% m-Na(‘l. IJH 8.5) medium to form the disrtlfide-bridged spwirs before rlrct~rophorcsis of thrsr fragmrnts on SDS-wnt,aining gels. Thus any segment of the mysin rod frrr to t1isswiat.t~ as a t.wo-chain unit in high-salt solution will. fiJIowing oxidation of it,s t,hiol groufw, tnigrat,r as a disllltidl~-l)r,itlg~(i dimrr on SI)S/f)olvac.rvlatnidr gel c~l~~c~trol)tiot’~~sis. Thv twwchain units of 1Ahl and short S-2 will consist, of it mixtjrrrc of’ ~OII(:ross-lillk~~d arid intramolr(:ular I)?ilS-cross-linked specirs. On the other hand. the slw(*ies tjhat have hecn cross-linked int~~J~tnolc~c:lllarl~ 1%ith I)MS \vill. follo\viny oxidation of tjht‘ir thiol groups. havr molwular \vrights higher t,han t,hr two-c-hail1 unit and \vill wnseqwntly he t,rapped at the tofw of the SIIS-containing gr4s. We weld. thwrforfb. wtlablish thck cross-linking rw.t.v const,:trlt.s of the mgosin srgmrnt.s to Ilrxighborillg tnolrculrs of t,he thick filamvnt, cwre l)y detwmining the ahsorhatlw of tht> two-clra,in units. I,%IiV and S-P, at various time int,wvwls. Figttrta I stio~vs tmiid l)a,ttertis of inyositl fragments derivrd from myofibrils produ&+ t,o form following vnzymat,ic c:leavrtg!r and oxidat,ion of the rrsulting dislllfid(l I)ridgvs. Both thr IAbI (AT, = S,j.OOO) and short S-2 (3~~ = 40.000) hands disappeared from thr SDS-containing gels \\ithin t’hrtv minut,t,s aRr>r initiation of oxidatiotk : this transformation \vas accompanied by tlw appearance of two IWU bands of n~olrc~ular weights - 130.000 and --UI .OW. wrresponding to thr disulfideThe al)sorl)ance of t’hew nw bridged LMM and short, S-2 specirs. rvsptvt~ively. I~ands lwcamt~ wnstatlt after three tninnt,rs and remained unchanged for lwriods 11~) reaction. wnsisttlnt with a quantjitatiw t,o three hours during the oxidat,ion disulfidr bridges arr cBollvc,rsiotl to t.hc tiisulfide-l)t~idgrd slwcitas. Although itltroduwd into shortj S-2 (as well as long S-2) by oxidation. \re find no disulfide l)ridgcs in thtl low molecular weight I,MiM t,hat is formed simultjaneously \vith short S-2 during t,he tryptic proteolysis step (SW iVat,erials and Methods). The HMM

(0) (b) (c) Cd) (e) Frc:. I, Elrc,trolrholPsis of myofibrillar protein with and without enzyme digestion in SDS-containing gels. .A Tris-glyc,inr buffer system was used awording to the method of Laemmli (1970) employing a stacking gel (W,, arrylamide. @lW,, methylene-biswr$amide) and a sqaration gel (75”,, acrylamidr O%P,, rnrthvlenr-bisacrlarni(l~). (a) C:lycerinated rabbit psoas myofibrils used in this study. (b) 1lyotibrils digested n-ith r-chgmotrypsin at pH A.5 and I = 0%. (c) Double-digested myofibrils. with 1 t,hymotrypsin and trypsin. (d) and (e) Oxidized samples of(b) and (c). respeectively. (a), (b) and (I,) Full! reduced by adding I”,, 2.mercaptoethanol to the samples loaded on the gel. JI, values. indic*at,ed on thr right of the Viguw. were determined in the phosphate buffer system according to the method of D’eber CyOsborn (1969) using myosin heavy vhain (210 x 103). HMM (150 x 103). LMM (75 x 103). actin (43 x IO’) short S 2 (10 x 103). and tropomyosin (33 x 103) as molecular weight standards. Lop .I/, ~T~II.Y mobilit>~IOPStxlt show a yw~l linearit,\- in the Tris-ylyc%w buffer syst,rm.

(heavy chain) band also disappears on oxidation. as does a major fraction of the actin I)and. Both of these species form high molecular weight oxidation products. which are trapped at the tops of the gels on electrophoresis. HMM and actin species are cross-linked. probably due to random intermolecular disulfide bridges in highsalt solution (I = 0%). The disulfidr>-bridged species were quantit,atirely reduced to the original chains by addition of d-mercaptoethanol.

628

H. ITENO

AXD

I\‘.

F. HARKIS(:‘I’OS

To ensure that the 130,000 and 91,000 M, bands originate from LMM and short S-2, respectively, these bands (Fig. l(d) and (e)) were cut out from the gels and then rerun on SDS-containing gels after treatment of the gels with excess Smercaptoet,hanol at pH 8.5 (Fig. 2). The 130.000 and 91,000 M, peptides migrated as 75,000 and 10.000 M, peptides. respectively. after reduction. When non-denaturing, pyrophosphate-containing polyacrylamide gels \vere uwd t’o analyze the enzymatic digestion and its oxidation product,s of myofibrils, thtl results shown in Figure 3 were obtained. Here. it will be seen, there is no detectable diffrrencr between the migration rat,es of myosin rods, LMM and short S-2 segments (derived from chymotryptic digestion of myofibrillar suspensions) and the disulfide-bridged species formed on oxidat)ion of these fragments in a high-salt (0% al-KC’I. pH 8.5) solvent. When isolated. purified fragments (rod, LSlSl and S-2) of myosin \ve.rt’ oxidized under the same ionic conditions. similar band patterns wrt obtained with no indication of high molecular weight, aggregates formed as a rvsultj of the oxidation procedure. Thus, it appears that specific int~ramolecula~ disulfidr bridges were introduced into LMM and S-2 segments and that the disulfidr oxidation technique allows us to distinguish between intramolecular and intermolecular cross-linking of myosin segments selectively following treatmwt \vith a bifunvtional cross-linking reagent.

(0) ,Yffwt of crowlitt kittg ott the hittdittg of stthfrag~rnettt-l to dirt

jilnmrnts

Earlier studies with dimethyl malonimidate (Sutoh B Harrington. 1977). which has only one methylene group between t,he t\vo imidate groups of the cross-linker.

75,000

-

40,000

(b) FIG:. 2. Ik~trophorcsis of myosin fragments in the reduced state in SDS-containing gels. Several hands (shown in Fig. 1) were cut out from the gels and then rwun in the phosphate buffer system accwdinp to the method of %‘eber M &born (19ti9). These protein bands were treated with excess 2mewaptoet~hanoi at pH %5 before electrtrphoresis. (a) SB x 103 band in Fig. I(b). (b) 130 x lo3 band in Fig. l(d). (c) 40 x 10’ band in Fig. l(c). (d) 91 x IO3 band in Fig. l(e). N, values are indicated on t,he ri,ght of the Figure.

(‘IlOSS-RIIIDGE

SH ss (a)

MOI’EIMEN’I

ASI)

SH ss

SH ss

MYOSIS

HP!)

HINGE

SH ss (d)

(cl

(b)

THE

SH SS (0)

FIG: 3. I-%+rophoresis of myosin fragments in the reduced (SH) or oxidized (SS) state in nondenaturing. p~rophosphate-containing gels. Myofibrils digested with enzymes ((a), (b), (c)) and purified myosin srgments (((I), (e)) were oxidized in the presence of Cu’+. Samples run on (a), (b) and (c) were t‘rac4ionatrd by the alcohol-denaturation procedure. Portions of the oxidized samples were reduced with vsc*ess %merc*aptoethanol before electrophoresis. Other details are described in Materials and Methods. (a) Myofibrils digested with a-chymotrypsin at pH 8.5. I = 0%: LMM. (b) Myofibrils double-dipestecl with r-chymotrypsin and trypsin. (c) Myofibrils digested with J-chymokypsin at pH 7.0. I = 0.1. in the pr~s~nw of E1)TA awording to the method of Weeds & Pope (1877): upper dense band is rod segment of mywin and lower faint one is tropomyosin. (d) LMM. (e) Low molecular weight S-2.

revealed no measurable effect of the cross-linking reaction on the binding of S-l to actin. It was important to determine whether cross-linking of rigor myofibrils with the longer cross-linker, DMS. would affect the actin-S-1 interaction. A mixture of actin -H-l complex (acto-S-l) and myosin rod filaments was prepared by digesting myofibrils with x-chymotrypsin in the presence of EDTA (5 mM) at I = 0.1 (pH 7.0) t.o clc~a.vet.hp junction between myosin S-l and rod segments (Weeds 8r Pope, 1977). This system was cross-linked with DMS for up to 30 minutes at 20°C”. Samples at, various stages of cross-linking were centrifuged to spin down actin filaments (or a&-S-I if S-l was bound) and rod filaments. The cross-linking reaction was monit,ored b,v densitometric measurements of the rod band on SDS-containing gels. Tht% results. shown in Figure 4. indicate that under these conditions up to 90% of thcb rod band disappeared due to cross-linking after 30 minutes of reaction, but, little cross-linking was observed between actin and S-l species. Supernatant fractions showed very little rod, S-l and actin on SDS-containing gels. indicating t,hat S-l \vas spun down due to binding to actin filaments irrespective of the stage of cross-linking. It’ appears from these studies that binding of myosin heads to actin filamrnt,s is not affected by DMS cross-linking of rigor myofibrils. (d) (‘roas-lindcing

of myqfihrils

with

dimethyl auhwimida.tu

The time-course of cross-linking LMM and the short S-2 segment of myosin t,o neighboring molecules within the thick filaments of rabbit myofibrils by DMS (pH 7.1) is presented in Figure 5. where the band intensities, which include non-

rod

(0)

(b)

(cl (d) (e)

(f)

(4)

(h)

( i 1

(I)

FI(:. 4. ~‘ross-linking of’ myufibrils n-ith DAIS. M~ofibrils. digested with x-c,hymotr\psin at I = 0.1, pH 7.0. in the presence of ED’l’.A (5 1m.1)to cleave the Iunction betwwn 5-I and rod segments. were cross-linked with I)MS (1.0 my/ml) for up to 30 min at 2OY’ and pH 7.0. LM~ofitxils at various stages of C~OSS~ linking KWP wntrlfut’r(l (lOO.(HK)g for 1 h). M@ihrils ((a) to (e)) and supernatant fractions after wntrifugation ((f) to (j)) were examined on SDS-oontaining gels according to thr mrthotl ul’ Laemmli (1970). using a gel consistillp of W,, acr~lamide and 0210,, meth~lene-hisac,rSlami~l~, (a) and (f) Mrofihrils without irdtlit ion 1>1’1)MS: c,rclss-linking time- :i min. (11) amI (g): 10 min. (c) and (h): 20 min. (d) anti (i). and 30 min. (e) and (j). Samples were treated with 2. rller~a~~tOt~ttlanol Irrii)rr rlrc,t r0ph0rmik.

Actm

S-l

Myosin

tj3P

H.

CTENO ASI)

\I’.

P HAKI
cross-linked and intramolecular (DMS) cross-linked species. show a tirst-order decay following electrophoresis on SDS-containing gels. This behavior was observed in our earlier studies on the cross-linking of S-l and rod in synthetic thick filaments and in glycerinated fibers and it was also seen in the present study of LMM and short S-2 at all pH values employed. The ratio of the rate constant, of cross-linking short. S-2 to that of LMM. kLy, (i.cl. is plotted w-s7~ pH in Figure 6. This normalized (aross-linking GdblM)). parameter eliminates the pH dependence of the amidination reaction and allows us

0.7

-9

,’

,/--

/ \ 0 II /\1’ ‘\ 0

0.6

i

0 “~:.~

l --*-0--

/’

---

\‘n I

I

7.2

8.0

7.6

8.4

PH

FIG. 6. Titrat.ion curves of the relative r&e of cross-linking (- l - l -) myosin short S-Z. I& ( =k, J kl,MM); and ( - - 0 - - 0 - - ) myosin heads, A$, ( = k, J/c,,). Cross-linking was carried out in 40 mMimidazole. HCI (pH 7.0 t,o 7-4) or in 40 miwtriethanolamin?~ HCI (pH 7.4 to 8.5) at 5°C. Cross-linking rate of myosin heads, k:,. from Chiao & Harrington (1979) and includes additional data collected in this study. kz., above pH 8.0 was slight,ly higher in this study than that described by Chiao & Herrington (1979). The broken line (- -- - -) indicates the relative cleavage rate at the HMM-LMM hinge region of the myosin heavy chains in myofibrils with x-chymotrypsin (see Fig. 9).

to compare the relative movement of the S-2 structure with that of the LMM segment, which is permanently immobilized within the thick filament core. On elevating the pH above neutrality, l& falls along a sigmoidal titration curve uer8u.s pH profile derived from similar to that observed earlier for the kz., (i&,/k,,) cross-linking studies of synthetic filaments (Sutoh et al., 1978a) and glycerinated 1979). ki., and kg., remain unchanged on muscle fibers (Chiao & Harrington, elevating the pH to about 7.4, but show a sharp decrease over a narrow pH range of their pH 7 values. (7.4 to 8.4), levelling off at about 30% and SO%, respectively, The results in Figure 6 strongly suggest outward movement of both the S-l and short S-2 segments from the thick filament surface on raising the pH above 7.4.

(‘ROSS-BRIDGE

(P)

fi’ffcxt of

pH

MOVEMENT

on the t-&r

of chymotryptic

A&D

THE

MITOSIS

clravnge

at the light

HINGE

(X3

meromyosin--henry

According to the helix-coil mechanism of muscle contract,ion (Harrington. 1971.1979: Tsong et al., 1979), a segment of the double-n-helical S-2 struct’ura. believed to be t)hr LMM--HMM hinge region. melts to random coil on release of the S-2 segment from t’he surface of the thick filament during a cross-bridge cycle. Thus we would expect this region of the myosin molecule to become more susceptible to the pH enzymat.ic proteolysis. With this idea in mind we ha.ve examined tlrpxdence of cleavage at the LMM-HMM junction in rigor myofibrils over the l)H is revealed by t,he cross-linking range in which release of the S-2 segment c,xperiments summarized in Figure 6. Myofibrils were digested with I-chymotrypsin (1 : 10. w/w) in dilute salt solutions buffered at different pH values over the range from 7.0 to 8%. Buffers always conto protect the head-rod linkage from cleavage by the enzyme tained (PI mnl-JIgzt (Weeds & Pope. 1977). When mgofibrils at 5°C were digested at pH 7.1, about 800/;, of the myosin heavy chains remained intact2 and very little HMM or LMM was formed after 50 minutes of digestion, as judged by densitometry of the banding pattern resulting from electrophoresis of this system on SDS-containing gels. However. w 85:/,)/,,of the heavy chains were cleaved at pH 8% (5°C) over the same time. resulting in rapid formation of LMM and HIMM as shown bp SDS/polyacrylamidr gel electrophoresis of the products (Fig, 7). The relative densit,y of the myosin heavy chain band on SDS-containing gels was determined at various digestion times and showed single exponential heharior when the log of the density of the

MYHC

1

Actin i

(a) L

(b) -

Cc) m

z- Bottom

FIN:. 7. z-(‘hymotrgptir digestion of myofibrils at pH 7.1 and 8%. Myofibrils were digested with I vh?(motrypsin (l-10. w/w. enzyme to protein) for 5Omin in %OmM-N&I, 0.1 mwMgC1,. 40mw imldazole. HCI (pH 7.1) or @l mM-MgC12. 40 mwtriethanolamine HCI (pH 8%) at 5°C. Digested material was analyzed with SDS-containing gels using the buffer system of Laemmli (1970) and a separation gel consisting of 8”;, acrylamide, 021 “lo methylene-bisacrylamide. Densitometrir recordings of the gels are shown. (a) Undigested m?ofibrils. (b) Myofihrils digested at pH 7.1: and (c) pH 8%. SMercaptoethanol was present in the dlpested sample. MyHC. myosin heavy chain.

634

H. I-EN0

ANI)

iv.

F. HAI~KTS(:‘l’OS

myosin heavy chain was plotted wrsus digestion time (Fig. 8). Formation of I,,\ilbl 1)~ digestion was detrrmined at) the same time. This process also showed singlr exponential bcahavior and \vas suprrimposal)lc on thr decay curve of thr myosin hca\-y chain band. The decay rate constant of t.hc myosin heavy chain. yHC. and the rate con&ant for t,hr formation of I,M?ul. Y,.~~. indicated cleavage I)g chymotrypsin at the LM&HMlCl junction. The rat)e con&ant at. pH 72 (5°C’. I = 0.03) was 0.32 +0.03 h ’ and tha,t at pH X.4 (5°C’. I = MM) was 2.1 +02 h - ’ giving an incrrase of six- to scvrnfold over this pH range. When t.hc digestions wcrv carried out in the presrncc of 0% ~Sa(‘l. in which myosin filaments are dissociated. the clravagc rate constant a,t pH 7.2 was 1.3 fO2 h- ’ and that at pH X.4 was 1.9 * 032 h ’

30 0

IO

20

30

40

DIgestIon time (mln) FIG:. 8. I)ipdion of mydihrils with vchymotrypsin at 6 (‘. hlyofibrils (I.0 mu protein/ml) were di#wtrd with w~hyrnotrypsirl (0.1 m@l) at pH 7.2 and 8.4. IIigestetl products were analyzed as tiwcrihetl in Materials and Methods. Relative dtvxsitiw of mywin heavy chain hands are plot~ted VPISUS 0.1 rn~-&$I,: digestion time. IXpestion conditions: (0 ) 40 mwimidazole HCY (pH 72). 10 mwXd‘l. (0) 40 mwtriethanolamine~ HC‘l (0) 40 rnwimidazolt~~ HCI (pH 7.2). 0% M-NaC’l. WI mkx.Mg(‘l,; (pH 8.4). @I mwM$l,: (m) 40 rnlM-triethanolnmin~. HC”l (pH 8.4). 0% rwNa(‘l. 0.1 m~-M$i~. The bars indic.ate the ranpc of rsprrimpntal error in nn;tl,vsis. -“-Mer~,aptoethHnol was present in the digested samplr~s;.

Since the intrinsic cleavage rate of S\-cllymotrypsin is clcvated 30?,, betwern pH 72 and X4 (Hess. 1971), the increase in cleavage rate in high salt solutions over this range is likclg to be the result of the pH dependencr of the enzyme rather than increa)srd susceptibility of the hinge region of myosin. It is interesting that the cleavage rate in dilute salt, solution at, pH 84, where myosiu is associated in t,hick filament,s. is comparable to that, observed in high salt solutions at this pH. This result suggests that, the conformational state of the myosin hinge region in the associated system. under ionic conditions where S-2 is released from t,he filament surface, is similar to that in dispersed myosin. On the other hand. mar neutral pH in dilut,e salt,. where thr S-2 link is down on the

(‘I:OSS.l-iKII)(:E:

MOVEMES’I

ASI)

THE

MFOSIS

HTN(:E

13.

filament surface. t hv cleavage rate constant is maximally one-quarter of its value in the dis~wrsrd system The rnt’e of chymotryptic cleavage at the LMM-HMM junction at 5”(‘, as measured I,- the rate of disappearance of the myosin heavy chain, is plotted as a function of pH in Figure 9. The rat)e constant. corrected for the intrinsic pH dependence of the enzyme. shows a sharp sigmoidal increase. wit,h the transition tnidpoint~ near pH 7.8. just over the pH span (see the broken line. Fig. 6) at which 5

4

0

thv cwss-linking rate of S-2 undcrgot~w a precipitous decline. and where we now Iwlic~ve t)hrt HhlM segment is released from the thick filament surface. The rate of frtrmat’ioll of HAlI and LM1/1 increased 1)~ a factor of about 8 over this range. +uggrst,ing either a transition within the hinge region to a. more open atructnre. \r.hich has incrcbased susceptibility to rnzymat)ic cleavage. or a change it1 accessibility of this region to the enzyme as the cross-bridge is released. It is worth twting that myofibrils at’ pH 8.5 (I = O-04) showed a clear A-band patt*ern under phasw~ont,rast microscopy. with no loss of any major protein after repeated washing 11y the solution. Purified LMM as well as rod segments are aggregated and spun down by ventrifugation (100,OOOg for 60 min) at 5°C. pH 8.5 and I = 0.04. Tt

636

H. t:ESO

seems reasonable

to assume that

ASI)

\\‘.

the thick

F. HARRISGTON

filament

system

remains

intact

in this

solverlt.

When the digestion was carried out, at a higher temperature (20”(r), the cleavage rate of the LNlZlGHMM junction gave a similar pH profile (Fig. 9). but in this case the relative rate of cleavage increased by a much larger factor. about ll-fold. over the pH range 7.0 to 8.6, again with the midpoint of the transition near 7.8. Since the concentration of I-chymotrypsin was reduced threefold (1 : 30, w/\v) in these experiments at high temperature, the rate of cleavage of the m;vofibrils at pH 8% (20°C’) \vas about six times that at 5°C‘. Taking into account the expected t’emperaturcl dependence of the rate of cleavage by chymotrypsin over this temperature interval ( -3.fold) it appears t)hat a larger amounts of substrat,e (large1 fract,ion of the LM&HM,Nl hinge) may be in the proteolyt,ically sensitive state at SOY as compared to PP. (f) Efffrct of trmpratvtrr

urd iorric strrrqth o/v the cross-linkivvg

rate of myosin brads

In view of the la.rge thermal dependence of the chymotryptic cleavage reaction. it’ M’as of interest to examine the effect of temperature on the cross-linking process. In these experiment)s \ve investigated changes in the cross-linking parameter kt., Since the variations in k:., parallel those in k&, t,he kc& values are expected to provide a measure of the outward movement of the HMM segment and thus of S-2 from thr thick filament surface. At neutral pH, raising the temperature from 5°C’ to 20°C had no significant effect on k:~, either at physiological (0.11 M) or low (0% M) ionic strength (Table I). showing that the HMM region lies close to the thick filament’ surface under all of these conditions. When the cross-linking was carried out at high pH (X.3 to 9.0) at physiological (0.11 RI) ionic strength, kz, showed a value of 015. which was higher than that measured at low (04.5 MM)ionic strength. That is, at

Prowli,rking

rate of myosin heads mndrr wa,riorcs conditiovvs. Effect of temperaturr ionic stwngth on ka I Temperature (“(‘)

Ionic strength? CM) oa5 0.1 1 w05 Wl:! CO5 Wll 0.05 0.11 0.11

k:,: 0.33 0.30 0.3% @31 @lo 0.15 @lo @Iti 0.13

t 40 mwimidazolr~ WC’1(pH 7.0 to 7.4) and 40 mwtriethanolamine~ HCI (pH X.3 to 90) were used, and ionic st,rength was adjusted with NaCI. $ Experimental errorinanalysisisabout lo”‘,, at pH 7.0to 7.4and 15”,, at pH 8.3 to 9.0.

a,nd

(‘!
MOVEIIIENT

,\SL)

THE

MYOSIS

HISGE

physiological (0.1 1 YI) ionic strength the A$~, wrsus pH plot exhibits sigmoidal profile than is observed at low (OG M) ionic st’rength.

635

a shallower

4. Discussion Thv sharp sigrnoidal decline in the relative rates of cross-linking. l& and A$.,. thr pH range 7.1 to 8.1 very probably originates from detachment and outward movement of the HMM segment of myosin from the thick filament surface. As \ve suggested earlier ((‘hiao & Harrington. 1979). this process appears t,o havtt a common origin in the synthetic filament system and in glycerinated psoas tibrrs in rigor. where a major fract,ion of the myosin heads are firmly attached to wtin (I,t~vcll & Harrington, 1980: (‘ooke 8; Franks. 1980). Plot’s of I& versus pH in both systems show a marked similarit)y in transition profile over this pH range. Wt helie\-cs the probable origin of the cross-bridge movement is electrostatic repulsion twt~wwn thtb cross-bridge and the thick filament surface. resulting from titration of ionizing groups, According to Rome’s (1967.1968) measurements of the equatorial reflwtions of low-angle X-ray diffraction patterns of glycerinated rabbit psoas muscle irnnwrsc~tl in dilut’e salt solution, the double-hexagonal lattice of thick and t,hin filaments tlxpands reversibly on elevating the pH. Xt an ionic st)rength t~quivalent~ t’o (I.10 RI, an expansion of the myosin t,o actin spacing of about 0.9 nm occurs ovw t,hr transition range (pH 74 to 84) observed in Figure 6. Lattice expansion also otwn-s on lowering the ionic strength and this is probably the reason for, thrl dwrrasc in cross-linking rat’e of the detached cross-bridges at pH 8.5 (sets Table 1 ). \\,hen the ionic strengt)h is lowered from 0.10 to OG. Rome (196X) obsrrvr~d an incarease of 1.0 nm in the myosin to actin spacing when the ionic strength was louvered over the same ionic strength range at pH 7 (,5”C). An even larger expansion is to be expected at pH 83. due to the higher net electrostatic thargtx on the filaments. The sharp I)H-dependent transition in proteolytic cleavage rate at the LMM HMXI hinge vould be explained by a shift in the steric accessibility of t,his region to the enzyme. ,4ltrrnatively. it could reflect a change in the conformation state of t,htl polypt~ptidr chains within the hinge segment. Thus. if the LMM-HMM hinge region is ordered in the double t-helical state when the S-2 link is bound to the thick filarnent~ surface (pH 7.0). the proteolytic cleavage rate should be markedly reduced just as it) is in the X~-helical, coiled-coil regions of the native LMM and short S-2 segmcwts of rnyosin. When the cross-bridge is released from the surface, partial mt~lting of the ‘vhelical structure within the hinge region would allow rapid clt~avagt~ by chyrnotrypsin. Although we cannot decide categorically bet,ween these two possibilities. at present we tend to favor the latter point of view for the following reasons: (1) at pH 8.5 only a small angular change in the orientation of the S-2 link is expected when the bridge is released from the thick filament surface : yet the cleavage of the hinge under these conditions is comparable to that of dispersed myosin molecules in high-salt solution. (2) At, neutral pH the rate of t~teavagt~ inweases about threefold when the temperature is increased from 5°C’ to 2O“C’. This increase approximates the t,ernperature dependence of the enzyme and suggests t’hat the conformational state of the hinge, when it is associated with t,he ovt~r’

634

H. I’ESO

ANI)

IV. F. HARRING’I’OS

filament surface, is unaffected I)?; temperature as it would be in the folded. X~-hrliral state. (3) Dissociation of myosin and rod minifilaments in pyrophosphate (5 rn3!) results in II decrease in k-helical caontJent,(ahout X4,, in rod at) room temperature). which is localized in t.hc S-2 region of rnyosin ((~riolLA~udit ut G(.. I981 ). The amount of the hinge strwturr in the unfolded (random roil) state is expectSed to increase with increasing t,emperature (Sutoh rt nl.. 19786). The increased cleavage rate of the LMST-HMM junction in t’hv released cross-bridge at 20°C’ compared to that. at 5°C’ (correctrd for the intrinsic t,emperature coefficient of the cnzymc) is consistent with a larger fraction of the hinge in its unfolded. proteolytically sensitive stat,c. The sirnilarit,.v in t,hr transition profilrs determined by cross-linking and by the protrolvtic kinetics (Fig. 6) suggests that the release process conforms to a twostate equilibrium between the associated and dissociated forms of the woss-bridge (see Tanford. 196X). Hence an appa,rent, equilibrium constant, can he defined as

is t,he fraction of bridges released frotn the surface at any pH value. From t,he general theory of linked functions (Wyman. 1964). the equilibrium between two states of a protein molecule that is dependent on pH may be expressed as WhW~f,,,

(2) where AZ represents the difference in the number of bound protons at any pH. When Kapp is calculated from the data points displayed in Figures 6 and 9 and log K appis plotted - ~KSIIS pH, a linear relat.ionship is observed over the transition region with slope (LIZ) of -2.1 +0.3 (Fig. IO). It appears that, the bridge release requires only a stnall change in local charge on the filament surface. a significant effc>c+of pH on the digestion of soluble Since we did not, olwrvt> ?,i\‘(.- :’ ;ZXmomt~r.i~~ myosin seems to have a similar conformation between pH 7.0 and 8.;;. The conformational transition of the polymerized myosin molecules, as detected by cross-linking and proteolgtic digestion met’hods, is probably caused b) a local shift in charge of some specific residues of the mvosin molecule. It seems reasonable to assume that such a local shift in charge could modify the apparent conformation of the molecules in t#he polymerized state through a co-operative interaction that, is absent in the monomeric state. The conformation of the cross-bridges in the rigor state at high pH (-8-S) is especially interesting, because the S-2 segment is free from the constraints of the thick filament backbone in contrast with the 9-2 segment at pH 7.0. According to the helix--random coil transition theory as described in earlier papers (Harrington, 1971 .I 979 : Tsong rf nl.. 1979). cross-bridges in t’he rigor state at pH 85 are expected to generate a force relative to those at pH 74. Our findings suggest. in accord with the hrlixPcoil transition theory of muscle contraction, that a glycerinated rigor fiber held at>constant length should generate

(‘ROSS-t’,t?Il)(:E

MOVERlEST

ANI)

THE

MMOSlN

HINGE

8 \’ ” i

IO

5

ti’N ..

0

OO

0

l

8

0

0

s

8

\

I

0

0

o-5 0

0 \ e

8 l ,

0.1 IL

,

\I 8

I

0

a wtravti\c forw on elevating the pH from 7 to 8.5 as a results of melting within this hinge region of the S-2 segment of myosin. It will lx of considerable- interest to dvtchrminr. ill futuw studies. whether such a prediction is fulfilled. ‘I’ht~ authors

art. grateful

to Dr Stephen

Lovrll

for suggesting

the S-S

bridging

c*x perimwts and for wry hrlpful discussions t,hroughout, the course of this work. M’tl also thatlk lb I’rt,cbr Knight for his rritical commrnt.s on the manuscript.. This investigation was

suplx~rtrd by I:nit,d States Public Health Service grant AMO4349. This is contribution IIO. IO96 from thv Department’ of Biology. iC1cCollum-Pratt’ Institute. The Johns Hopkins I’nirtlrsity. Baltimore. Mel 21218. U.S.A. This research was carried out, during the tenure of a I’ost -doc%oral

Ft~llowship

(to H L’.) from

(‘hiao. Y. (‘. & Hitrrington, W. (‘ookr. R. & Franks. K. (1980). Harrington. W. F. (1971). Pror. Hiwrington. LV. F‘. (1979). I’ ror.

the Muscular

Dystrophy

.Asso&dion.

F. (1979). Hiochemistry, 18. 959-963. Hiorhmt.istry, 19, 2265 2268. Sat. =Icnd. Sri.. l’.S..-l. 68, 685~689. Sot. Accld. SC+.. P.S..4 76, .X%6--5070

ti40

H. I’E:NC)

ANI)

\2’. V’. HAK.KIN(:‘I’OS

Hess. (:. I’. (1971 ). In Thr Enzymea (Hoyer, I’. I).. ed.), vol. 3, pp. 213-246, Academic Press. Sew York. Huntrr. RI. J. B Ludwig, AI. L. (1962). J. --4wlrr. (‘hjum. Sot. 84, 3491-3504. Laemmli. Cl. K. (1970). AVaturr (Lorubn), 277, 6X0-685. Lovell. S. ,I. & Harrington, W. F. (1980). Frd. Proc. Fud. .-im~r. ivot. Exp. Biol. 39. 193.i. Oriol-Audit. C’., Lake. ,l. A. & Reisler, E. (1981). Riochernistry, 20. 679-686. Rome, E. (1967). J. Mol. Riol. 27, 591-602. Rome, E. (1968). ./. Mol. Biol. 37, 331-344. Stewart. 53. (1975). FEBA’ Lrttws, 53, 5 7. Sutoh. K. K: Barrington. LV. F. (1977). Riochr,mi.stry. 16, 2441-2449. Sutoh. K.. (‘hiao, Y. (‘. & Harringt,on. W. F. (197%). Biochemistry, 17, 1234.-1239. Sutoh. K.. Sut,oh. K.. Karr. T. Ps Warrington. W. F. (197%). J. Mol. Biol. 126. I-22. Cent,-(:yiirggi. A. (i.. (‘ohen. (‘. & I’hilpott. D. E. (1960). J. 1Mol. Biol. 2, 133 ~142. Tanford. (‘. (1968). .-Idvnnces iu f’rotri,c Phwmistry. vol. 23, pp. 121-282. Academic Press. Nrw \;ork. Tsong. T. \-., Karr. ‘I’. & Harrington. W. P. (1979). f’roc. Snf. ilcad. AX.. r’.S.-1. 76. IlOg11 13. IVeber, K. K- Osborn. &I. (1969). J. Hl,ol. Chrm. 244. 44MS412. Weeds, A. (i. & Pope, B. (1977). J. Mol. Biol. 111. 129-157. b+‘ynan. .J. (1964). .-1dvnuc~,s in P rofuin Chemistry. vol. 19. pp. 223-286, Arademic Press. Srw York.