doi:10.1006/jmbi.2001.4490 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 307, 137±147

Calcium-dependent Structural Changes in ScallopHeavy Meromyosin

W.F. Stafford1, M.P. Jacobsen1, J. Woodhead2, R. Craig2

E. O'Neall-Hennessey3 and A.G. Szent-GyoÈ rgyi3*

1Boston Biomedical ResearchInstitute, WatertownMA 02472, USA2Department of Cell BiologyUniversity of MassachusettsMedical School, WorcesterMA 01655, USA3Department of Biology andRosenstiel Biomedical ResearchCenter, Brandeis UniversityWaltham, MA 02254, USA

E-mail address of the correspondszentgyorgyi@Brandeis.edu

Abbreviations used: HMM, heavysubfragment-1; S2, subfragment-2; Rchain; ELC, essential light chain; EGbis(baminoethyl ether)N,N,N0,N0-tetAMPPNP, 50-adenylyl-b,g-imidodip

0022-2836/01/010137±11 $35.00/0

The mechanism of calcium regulation of scallop myosin is not under-stood, although it is known that both myosin heads are required. Wehave explored possible interactions between the heads of heavy mero-myosin (HMM) in the presence and absence of calcium and nucleotidesby sedimentation and electron microscope studies. The ATPase activityof the HMM preparation was activated over tenfold by calcium, indicat-ing that the preparation contained mostly regulated molecules. In thepresence of ADP or ATP analogs, calcium increased the asymmetry ofthe HMM molecule as judged by its slower sedimentation velocity com-pared with that in EGTA. In the absence of nucleotide the asymmetrywas high even in EGTA. The shift in sedimentation occurred with asharp midpoint at a calcium level of about 0.5 mM. Sedimentation of sub-fragment 1 was not dependent on calcium or on nucleotides. Modelingaccounted for the observed sedimentation behavior by assuming thatboth HMM heads bent toward the tail in the absence of calcium, while inits presence the heads had random positions. The sedimentation patternshowed a single peak at all calcium concentrations, indicating equili-bration between the two forms with a t1/2 less than 70 seconds. Electronmicrographs of crosslinked, rotary shadowed specimens indicated that81 % of HMM molecules in the presence of nucleotide had both headspointing back towards the tail in the absence of calcium, as comparedwith 41 % in its presence. This is consistent with the sedimentation data.We conclude that in the ``off'' state, scallop myosin heads interact witheach other, forming a rigid structure with low ATPase activity. Whenmolecules are switched ``on'' by binding of calcium, communicationbetween the heads is lost, allowing them to ¯ex randomly about thejunction with the tail; this could facilitate their interaction with actin incontracting muscle.

# 2001 Academic Press

Keywords: scallop; heavy meromyosin; structure; sedimentation; electronmicroscopy

*Corresponding author

Introduction

Myosin-linked regulation of muscle contractionoccurs by two different mechanisms: molluscanmyosins are activated by direct calcium binding,1,2

while smooth and non-muscle myosins are acti-

ing author:

meromyosin; S1,LC, regulatory lightTA, ethylene glycol-

raacetic acid;hosphate.

vated by phosphorylation.3 ± 5 Despite the differentroutes of activation, regulated myosins have simi-lar features. Both types can exist in vitro in a 10 Sform where the rod portion folds back and takesup a ``safety pin'' con®guration in the ``off''state.6 ± 9 The two-headed soluble fragment of myo-sin, heavy meromyosin (HMM), also shows a shifttoward a con®guration where the heads bend backtoward the rod in the unphosphorylated10 orcalcium-free state.11

Calcium-dependent regulation of scallop myosinand myosin fragments requires the presence ofboth myosin heads. In contrast to myosin andHMM, which have a very low ATPase activity

# 2001 Academic Press

Figure 1. Two populations of HMM are produced byshort tryptic digestion. SDS/7.5 % polyacrylamide gels.(a) Unfractionated HMM. (b) Enriched 156 kDa HMM.(c) Enriched 141 kDa HMM. (d) Unfractionated HMMon SDS/15 % polyacrylamide gels.

138 Changes in Scallop Heavy Meromyosin by Calcium

without calcium, single myosin heads (subfrag-ment 1, or S1) are fully active in its absence.12,13

Calcium binding by myosin and HMM becomescooperative in the presence of nucleotides, and cal-cium weakens nucleotide binding.14 Communi-cation between calcium and nucleotide-bindingsites operates only in two-headed molecules,although both sites are present and functional insingle heads. The ability to communicate betweenthese sites is a precondition of the off state where,in the absence of calcium, entry to the contractilecycle is somehow prevented. However, the uniquestructure of this off state has not yet been deter-mined.

Here, we analyze nucleotide and calcium-dependent structural changes in scallop HMM bysedimentation and electron microscopic studies.Scallop HMM is well suited for such studies, sincethe calcium switch operates at low, close to physio-logical, calcium concentrations. This work extendsan earlier study which used electron microscopyand proteolytic susceptibility to demonstrate aquantitatively small calcium-induced shift in headorientation in scallop HMM.11 Our use here of anew, highly active and sensitive HMM prep-aration, and of sedimentation velocity as an ana-lytical tool, leads to new, physiologically relevantinsights into the regulation of scallop myosin. Ourmodeling of the sedimentation pattern indicatesthat the two heads of HMM bend toward the tailin the absence of calcium, provided ATP analogsor ADP are present, while the heads are randomlyoriented in the presence of calcium or in theabsence of nucleotides. Electron microscopy ofcrosslinked HMM molecules provides images thatare consistent with this model. Sedimentationstudies show that the two HMM conformations arein equilibrium. This work was presented in apreliminary form at the 43rd Annual Meeting ofthe Biophysical Society.15

Results

Characterization of HMM preparations

Digestion of scallop myosin produces HMMwith high activity (0.85(�0.15) sÿ1 turnover ratein the presence of calcium without actin) andover 90 % calcium sensitivity {(1 ÿ ATPaseEGTA/ATPaseCa)X100}. The HMM had two heavy chainbands corresponding to putative chain weights of156 kDa and 141 kDa in a ratio of 60 to 40 %.Weak additional bands with sizes close to intactmyosin heavy chains and some breakdown pro-ducts of about 50 and 70 kDa are also present(Figure 1). The higher molecular weight com-ponents could be eliminated by ammonium sulfateat 50 % saturation. After removal of the soybeantrypsin inhibitor the only low molecular weightbands seen are those of the light chains, at17.5 kDa (Figure 1).

The absence of 23 kDa and 20 kDa fragmentsobtained after more extensive tryptic digestion16

indicates that the difference between the two majorbands is not due to motor domain degradation(Figure 1). Proteolysis of the motor domain is lim-ited by the very short tryptic digestion used. Moreprolonged digestion increases the concentration ofthe lower molecular weight HMM but also leads toproteolysis of the motor domain and a decrease incalcium sensitivity. Because the motor domainremains intact, the weight difference between thetwo heavy chain bands must represent a differencein tail length. The difference corresponds to anapproximately 20 nm long coiled coil a-helix. Thetwo HMMs could be partially separated on Sepha-rose 4B or Sephacryl acrylamide 500 columns.Based on the ratios of the 156 and 141 HMM kDachains the higher molecular weight HMM could beenriched to 90 % purity, the lower molecularweight HMM to 70-80 % purity at the present stageof puri®cation (Figure 1).

Electron microscopy reveals that the HMM mol-ecules produced by the very short tryptic digestionfall into two classes with mean tail lengths ofabout 69 and 47 nm (Figure 2). The ratio of thelong to short HMMs agrees well with the estimatesobtained from densitometry of the gels both for theunfractionated and fractionated preparations. Thetails of the long HMM molecules were frequentlybent about 22 nm from the end, indicating the pre-sence of a ¯exible hinge region, which appears tobe close to the proteolytic site for the shorterHMM. Therefore, there are two populations ofHMM molecules arising from tryptic hydrolysis oftwo nearly equally available sites of the myosinrod that are attacked before the motor domain isnicked signi®cantly. Very long molecules havingtail lengths of around 100 nm are also occasionallypresent, probably corresponding to the smallamount of higher molecular weight componentobserved on the gels.

Figure 2. The two populations of HMM differ in theirtail lengths. (a) to (c) Electron micrographs of rotaryshadowed HMM. (a) Long HMM with the tail fullyextended. (b) Long HMM exhibiting a bend in the tailapproximately 22 nm from the C-terminal end, corre-sponding to the tail length difference between long andshort HMM, indicating ¯exibility at this site. (c) ShortHMM. The scale bar represents 20 nm. ((d) to (f)) Histo-grams of HMM tail length distribution from electronmicroscopy. (d) Unfractionated HMM. (e) Enriched longHMM preparation. (f) Enriched short HMM preparation.

Changes in Scallop Heavy Meromyosin by Calcium 139

Effect of calcium and nucleotideson sedimentation

The sedimentation pro®le of the long HMMshows a single unimodal boundary at around 8 Sin the presence of calcium and at around 9 S in itsabsence, consistent with the presence of a singlemolecular species. The HMM preparation that hadnot been fractionated also sedimented as a singlepeak, both in the presence and absence of calcium;however, its patterns are somewhat broader thanwould be expected from a single component. Thelong HMM sediments somewhat faster (by about4 %) than unfractionated HMM due to the bendingof the tail. A pliable region in the tail of the longHMM is indicated by the bending of the C-term-inal segment (Figures 2 and 5). Modeling of longHMM indicates that the increase of mass combinedwith the ¯exibility of the C-terminal 22 nm of thetail results in a higher sedimentation coef®cientbecause the frictional ratios of both long and shortHMM are then similar.

HMM complexed with ADP sediments faster inthe absence of calcium than in its presence, indicat-ing that in EGTA the structure of HMM is morecompact (Figure 3, Table 1). Both fractionated andunfractionated preparations exhibit the same cal-cium effect on sedimentation, although the actual Svalues for unfractionated preparations are some-what lower. The calcium-dependent shift in sedi-mentation coeff®cient, measured on long HMM,has a midpoint at about 0.5 mM calcium and a Hillcoef®cient of 1.63 (Figure 4), close to the valuesobtained previously for calcium binding in the pre-sence of ADP.14 All the sedimentation patternsobtained at different calcium concentrations showsingle peaks. Therefore, the two different ADPbound structures obtained in the presence and inthe absence of calcium are in rapid equilibrium onthe time scale of sedimentation. Resolution of con-formers would require a half-life greater than70 seconds to be detected. The extreme values ofs20,w obtained from the pCa titration curve were9.08 S and 8.02 S in EGTA and calcium, respect-ively. It was possible to model these values byassuming that the heads were fully back againstthe tail in the presence of EGTA and by assuminga fully random distribution of head orientations inthe presence of calcium (Figure 5(a) and (b)). Themodeling gave values of 9.06 S and 8.11 S for thesestructures.

ATP and various ATP analogs (includingAMPPNP, MgADP.Vi, MgADP.BeFx, MgAD-P.AlF4) can substitute for ADP in supporting acalcium-induced change in sedimentation (Table 1).In contrast, in the absence of nucleotides, removalof calcium does not increase the sedimentationrate, indicating that HMM does not adopt themore compact structure unless nucleotide is pre-sent. Sedimentation of S1 is independent of cal-cium and of nucleotides.

Table 1. The calcium effect requires both heads and the presence of nucleotides

Protein Analog s* � Ca2 � s* ÿ Ca2 � �s*/s*a

S1 MgADP 5.85 5.85 0.000HMM None (rigor) 7.50 7.70 ÿ0.026HMM (long) MgADP 8.16 9.05 ÿ0.103HMM MgADP 7.82 8.70 ÿ0.107HMM MgATP 7.60 8.30 ÿ0.088HMM MgAMPPNP 7.64 8.32 ÿ0.085HMM AlF4.MgADP 8.15 8.95 ÿ0.094HMM Vi.MgADP 7.90 9.05 ÿ0.135HMM BeFx.MgADP 7.95 9.10 ÿ0.135

All data shown in this Table were obtained on preparations that were mixtures of long and short HMM except for the one labeledHMM(long) which was about 90 % long HMM. Note that long HMM sediments faster than short HMM because of the bending ofthe tail.

a �s*/s* is the fractional change in s* and is expressed as the ratio to compensate for variations in the composition of HMM fromone preparation to the next. This represents the change on going from EGTA to calcium.

140 Changes in Scallop Heavy Meromyosin by Calcium

Electron microscopy

The heads of scallop HMM observed by rotaryshadowing can adopt a variety of angles withrespect to the tail (Figure 6). The head dispositionsof uncrosslinked HMMs are presented as classi®edby Suzuki et al. for gizzard HMM10 (Table 2). Inthe presence of calcium and ADP there is anincrease in Type 1 (``heads up'') and a decrease inType 2 (``heads out'') and Type 3 (``heads down'')con®gurations when compared with the low cal-cium state. The changes observed are relativelysmall, and a considerable number of HMM mol-ecules appear to remain in the heads up positioneven in the absence of calcium. However, we haveobserved that there were often patches on the micasubstrate on which the molecules are depositedwhere the majority of the HMM molecules were ina heads down conformation in the absence of cal-cium. This uneven distribution may have beencaused by variations in the interaction of the HMMmolecules with the mica, suggesting that thecharged mica surface can signi®cantly affect thedisposition of the heads, possibly masking thestructural changes in solution implied by the sedi-mentation data.

Because of this possible substrate effect, we havealso examined molecules whose conformation hasbeen ®xed by crosslinking in solution with 0.3 %glutaraldehyde for one hour at 4 �C before apply-

Table 2. Calcium dependence of head orientation by electron

Native HM

Class Conformationb �Ca2 �

1 ``Heads up'' 502 ``Heads out'' 303 ``Heads down'' 12

a HMM was ®xed with 0.3 % glutaraldehyde for one hour inessentially complete crosslinking before application to the mica.10

b Head orientations were classi®ed into three groups.10 Crosslinktions.

ing to the mica surface. In this case a different dis-tribution of heads up and heads down molecules isobtained. Under these conditions, the heads of allthe HMM molecules were crosslinked in either an``up'' or ``down'' position with no heads orientedin an ``out'' position. In addition, the 22 nm longstretch at the C-terminal end of the tail of longHMM cross-linked in a folded conformation,resulting in an apparent tail length very similar tothat of short HMM. In the heads down position,the 18 nm long heads appeared to have cross-linked with each other and also with the N-term-inal end of the tail, resulting in an apparent taillength for both short and long HMM that wasabout 18 nm shorter than for the heads up pos-ition. Therefore, the apparent tail length of bothlong and short HMM in the heads up and downpositions was approximately 47 nm and 29 nm,respectively, giving a readily observable difference.This was important because the crosslinkingusually made the orientation of the heads dif®cultto discern. Separation into up or down classes wasstraightforward, however, on the basis of apparenttail length (Figure 6).

The proportion of molecules in the ``headsdown'' orientation increases signi®cantly to 81 %(compared to 17 % for native HMM) when cross-linking takes place in calcium-free medium in thepresence of ADP, with only 19 % of the headsoriented in the up position. When crosslinking

microscopy in the presence of ADP

M (%) Crosslinked HMMa (%)

ÿCa2 � �Ca2 � ÿCa2 �

39 59 194417 41 81

the presence of ADP with and without calcium, resulting in

ed HMM shows only ``heads up'' or ``heads down'' conforma-

Figure 4. Dependence of sedimentation on calciumconcentration. Sedimentation coef®cients versus free cal-cium concentrations. Existence of a single peak at eachcalcium concentration indicates a rapid equilibrium (onthe time-scale of sedimentation) between the calcium-bound and calcium-free states. Midpoint of transition isat pCa 6.33 with a Hill coef®cient of 1.63. Sedimentationof 0.3 mg/ml HMM in 80 mM NaCl, 2 mM MgCl2,20 mM Mops, 0.5 mM ADP, 0.5 mM DTT, 3 mM NaN3,1 mM EGTA with varying CaCl2 at 20 �C and pH 6.8.

Figure 3. Column puri®ed ``long'' HMM sediments asa single boundary at all calcium concentrations. Themain peak gives an apparent molar mass at 373 kDawhose sedimentation coef®cient depends on free cal-cium. Curve A, pCa 4.57; curve B, pCa 6.44; curve C,pCa 7.28.

Changes in Scallop Heavy Meromyosin by Calcium 141

occurs in the presence of calcium, 59 % of the mol-ecules have their heads up and only 41 % down(Table 2).

Discussion

Using the complementary techniques of sedi-mentation and electron microscopy we haveshown that the structure of the off state of scallopstriated muscle myosin involves a folding back ofthe myosin heads towards the tail, and that switch-ing on is associated with a loosening of this struc-ture, allowing the heads to move away from thisposition.

Properties of scallop HMM

Two sites of scallop myosin are rapidly attackedby trypsin to produce a short and long HMM. Elec-tron microscopy shows that the two populations ofHMM differ in tail length by about 22 nm. Theapproximately 15 kDa difference in the heavychain peptides is close to the expected weightdifference of a 20 nm long a-helix. The sites of theearly tryptic hydrolysis seem to be in the generalregion of the hinge where myosin tails appear tobe ¯exible17 ± 19 and melt at elevatedtemperatures.20 ± 22

The very low ATPase activity of the HMM prep-aration in the absence of calcium, and the tenfoldto 15-fold activation in its presence indicates thatover 90 % of the molecules are regulated; i.e. in theabsence of calcium they cannot participate incontraction. An HMM preparation consistingmostly of regulated molecules has also beenobtained previously in limited amounts by rapidcentrifugation in an airfuge in the presence of ATPand actin.23

Nucleotide requirements for the ``off'' state

In the absence of calcium and in the presence ofATP-regulated myosins and HMMs are in the offstate and cannot enter or complete the ATPasecycle. Both cooperativity and sedimentation studiesindicate that the structure of the off state is notspeci®c to ATP, since a similar structure can beobtained by a number of different nucleotides.Therefore, the off state is clearly different from anyof the states of the contractile cycle. The differentmyosin states in the cycle can be made static andvisualized with the aid of particular ATP ana-logs.24 ± 28 These include two ATP states, an ADP.Pi

state and possibly a nucleotide-free, actin-boundstate, corresponding to the 90 �, 45 � and 15 � pos-itions of the lever arm. In contrast to the speci®crole of a particular ATP analog to stabilize thesestatic states, the off state is obtained by most of thenucleotide analogs. The lack of requirement for aspeci®c analog can be seen both in the cooperativ-ity between the two heads and the higher sedimen-tation rate. Most studies on the nucleotiderequirement for the off state of smooth muscleHMM or myosin have been restricted to ATP. It isnoteworthy, however, that AMP.PNP had thesame effect as ATP on the sedimentation of chickengizzard HMM;10 therefore, the lack of speci®city ofthe ATP analogs to stabilize the off state may beoperative in both regulatory systems.

Figure 5. Hydrodynamic modeling of long HMM. Inthe absence of calcium the model indicates that allheads must be in the down position; in its presence arandom distribution of the heads can account for themeasured sedimentation. Two factors were taken intoaccount in the modeling: head orientation and tail bend-ing: The observed sedimentation behavior could beexplained by the above set of models using the programHYDRO.56 The random tail orientations that were usedfor all the models are indicated. The bending of the tailexplains why long HMM sediments a little faster thanshort HMM. The extreme model with the heads bentdown against the tail could represent a cooperativeinteraction between the heads in the ``off'' (calcium free)state. Modeling of this state yields an unambiguoussedimentation value of 9.06 S. The value of 8.02 S forthe ``on'' state (calcium saturated) was obtained as anaverage of all head conformations as described inMaterials and Methods.

Figure 6. Calcium dependence of head orientationdetermined by electron microscopy of rotary shadowedHMM in the presence of ADP. (a) to (c) Examples ofuncross-linked HMM adsorbed onto mica47 and classi-®ed into (a) Class 1 (``heads up''), (b) Class 2 (``headsout'') or (c) Class 3 (``heads down'') conformations.10

(d) and (e) Examples of HMM crosslinked with glutaral-dehyde in solution at 4 �C and sprayed onto mica.11

HMM was crosslinked either in the (d) ``heads up'' con-formation or (e) ``heads down'' conformation. LongHMM is on the left and short HMM is on the right ofeach pair of examples. Note that in crosslinked prep-arations, the end of the tail is at a site corresponding tothe hinge region, resulting in similar tail lengths forboth long and short HMM molecules. In long HMM, thefolded region often appears as a thickening of the tail.The ``up'' and ``down'' position of the heads can be bestdetermined from the apparent tail lengths. The differ-ence between these orientations corresponds to taillengths of approximately 47 nm and 29 nm, respect-ively. The scale bar represents 20 nm.

142 Changes in Scallop Heavy Meromyosin by Calcium

Analysis of sedimentation patterns andelectron microscopy

There appears to be no preferred angle of orien-tation between the two heads, or the heads and thetail, for either rabbit skeletal myosin17,18 or scallopmyosin29 examined by electron microscopy underhigh salt conditions. This suggests that the headsare capable of swinging freely about the head-tailjunction. There is also evidence that myosin headscan rotate independently around their longaxis.30,31 Modeling the sedimentation patterns ofboth the unfractionated (not shown) and longHMM preparations shows that at physiologicalionic strength both heads of most of the moleculesmust bend back towards the tail in the off state, i.e.with ADP and in the absence of calcium (Figure 5).The heads could be immobilized in this state byinteracting with the rod and possibly with eachother. In contrast, in the presence of calcium thesedimentation constant ®ts a model in which theheads can take up random positions. This couldoccur if calcium breaks the interactions betweenthe two heads or the heads and the tail, resultingin a structure that allows the heads to undergo thechanges required for contraction. The Hill coef®-cient of 1.63 for the structural change agrees withthe cooperativity of calcium binding in the pre-

sence of ADP.14 This interpretation of the sedimen-tation data is consistent with observations onnative and synthetic scallop myosin ®lamentswhich show that an orderly arrangement of theheads at the surface of the ®laments under relaxingconditions becomes disordered in the presence ofcalcium.32,33 A head-to-head interaction in the offstate is also implied by the inability of S1 andsingle-headed myosin to achieve this state, andfrom cooperativity studies that demonstrate thattwo heads are necessary for communicationbetween the nucleotide and the calcium bindingsites.14

A shift toward the heads down morphology ofHMM when calcium is removed is also indicatedin the electron microscopy images of native mol-ecules adsorbed onto a mica surface (Table 2).However, this shift is only partial and a signi®cantportion of HMM molecules appear to remain inthe heads up position in the absence of calcium, asdescribed by Frado & Craig.11 Since the prep-aration is over 90 % sensitive, one would expect

Changes in Scallop Heavy Meromyosin by Calcium 143

that a similar percentage of molecules shouldassume a head orientation characteristic of the offstate. It appears that the non-covalent interactionsmaintaining the off state are adversely affected bypreparation of the samples for rotary shadowing.This is suggested by the ®nding of patches on thegrids where most of the heads bend towardthe rod presumably resulting from variations inthe electrostatic properties of the highly negativelycharged mica surface. This interpretation is sup-ported by the results of crosslinking studies. Inthese experiments, HMM was fully crosslinkedwith low concentrations of glutaraldehyde in sol-ution before being applied to the mica. For cross-linking to occur, the two heads must be in closeproximity in either the heads up or the headsdown conformation. The ratio between these twocrosslinked states will be proportional to the timespent in the respective positions. In the absence ofcalcium 81 % of the HMM molecules showedheads down structures; and this was reduced to41 % by calcium. The large effect of calcium on theHMM head disposition indicated by these cross-linking studies is in good agreement with the sedi-mentation studies. The signi®cant fraction of HMMmolecules remaining in the heads down confor-mation in the presence of calcium may re¯ect therandom ¯exing of the heads in high calciumsuggested by the sedimentation modeling. If thiswere completely random, then 50 % of the mol-ecules would be expected to be crosslinked in thisstate in calcium. The large perturbation in headdisposition of uncrosslinked molecules that wehave observed at low calcium, apparently causedby the mica surface, may partially explain earlierobservations of a smaller than expected number ofmolecules with heads down.10,11 It suggests thatconsiderable caution should be exercised in inter-preting conformations of ¯exible molecules usingthe mica shadowing technique.

The calcium-induced shift in sedimentation isconsiderably reduced at 4 �C (data not shown) andamounts to about half of the change of sedimen-tation coef®cient obtained at 20 �C. In contrast, cal-cium sensitivity of the ATPase activity of myosinremains unaltered at 4 �C;34 the same is true forHMM (data not shown). A temperature-dependentchange in the ¯exibility of the head-rod junctionmay account for this disparity. Future studiesinvolving electron microscopy of native frozenhydrated HMM molecules or of 2D HMM crystal-s35 may provide an unequivocal visualization ofhead orientations in the off state.

Communication between the heads in HMM

A direct interaction between the two heads in anoff state HMM has been suggested recently by elec-tron microscopy of two-dimensional crystals ofunphosphorylated gizzard HMM.35 In this struc-ture the position of the two heads is asymmetric,the actin-binding interface of one head being incontact with the converter region36 of the other. It

is proposed that the structure may inhibit ATPaseactivity by preventing domain motion necessaryfor phosphate release. An interaction between thetwo heads of scallop HMM is also indicated by the1.63 Hill coef®cient of calcium activation and bythe calcium dependence of sedimentation. This issupported by electron microscopy of shadowed,crosslinked molecules. It is likely that the structureof scallop HMM in the absence of calcium will besimilar to the unphosphorylated form of smoothmuscle HMM. In both systems, sedimentation datashow that the off state has a lower asymmetry. Thereduced asymmetry of HMM in the off state canonly be modeled by the bending of the headstowards the rod. Thus, while the rod was notresolved in the crystal studies, our results implythat in the off state the heads interact not onlywith each other but also with the rod. Molluscanand gizzard RLCs of the two myosin heads can becrosslinked at sites close to the head-rod junctionboth in the absence of nucleotides37,38 and in theoff state.39 ± 41 It has been proposed that a particularrod sequence of the ®rst heptad following theinvariant proline is a prerequisite for regulation ofrecombinant chicken gizzard HMM;42 however,deletion mutants suggest that speci®c residues atthe head-rod junction may not be necessary.43 Flu-orescence studies indicate that the presence of therod alters the af®nity to the RLC.44 It still remainsto be shown how the RLCs act as inhibitory sub-units and how they contribute to the stabilizationof such a structure.

Relevance to contractile activation in vivo

The conclusions from this work are qualitativelysimilar to those from an earlier study which usedelectron microscopy and proteolytic susceptibilityto demonstrate a calcium-induced shift in orien-tation of a small fraction of the heads of scallopHMM.11 This study used HMM with only moder-ate calcium sensitivity, and did not provide aquantitative assessment of HMM conformation insolution. Our sedimentation study of a highlyactive and sensitive HMM preparation demon-strates a quantitative transformation in HMM con-formation in solution between low and highcalcium states, and this is supported by electronmicroscopy of crosslinked molecules. Analysis ofthe sedimentation patterns indicates that the twostructures equilibrate with a half-life of less than70 seconds. However, the time-scale of sedimen-tation is not short enough to demonstrate whetherthe change occurs at a physiologically relevantrate. Scallop striated muscle is activated rapidlyand reaches peak tension within 90 milliseconds.45

The binding of calcium and the release of ADPoccur at speeds compatible with the physiologicaldata.13,46 The change in the myosin heads that wehave observed, from a state of limited mobility inthe absence of calcium to a more ¯exible structurewhen calcium is bound, could readily account forthe disordering of myosin heads that occurs when

144 Changes in Scallop Heavy Meromyosin by Calcium

scallop myosin ®laments are activated bycalcium.32,33 The transition is rapid (<40 ms; R.Craig, unpublished observations) and thereforelikelyto be physiologically relevant. The existence ofmyosin-linked regulation in a wide variety ofvertebrate and invertebrate muscles2 suggests thatour results may have broad signi®cance in theunderstanding of muscle activation.

Materials and Methods

Protein preparation

S1 was prepared from striated scallop muscle myosin(Argopecten irradians) as described by Kalabokis & Szent-GyoÈrgyi,14 with the following modi®cations: using a1/2500 papain to myosin weight ratio; eight minutesdigestion at 20 �C; and including 0.5 mM ADP for theReactive red chromatography. HMM was prepared from4-6 g of myosin (15 mg/ml) which was digested withtrypsin (2.5 units/mg myosin) in 0.5 M NaCl, 5 mMMgCl2, 0.5 mM CaCl2, 10 mM Pi, 10 mM Mops, 0.5 mMDTT, pH 6.8 for 2.5 minutes at 20 �C. The very limitedtryptic digestion produced highly active and calcium-sensitive HMM (although at low yield) in which themotor domain remained intact, showing very little nick-ing. The digestion was stopped by the addition of soy-bean trypsin inhibitor at 10 mg/mg trypsin, followed bydialysis against 40 mM NaCl, 1 mM MgCl2, 0.1 mMEDTA, 3 mM NaN3, 0.5 mM DTT, 5 mM Pi (pH 7). Thesample was clari®ed by centrifugation at 100,000 g for30 minutes, 5 mM diadenosine pentaphosphate wasadded to the supernatant to inactivate possible adenylatekinase contamination. HMM was precipitated by adding1.22-1.5 volumes of cold saturated ammonium sulfatesolution to a ®nal 60 % saturation. The pellet was dis-solved in a small volume of 80 mM NaCl, 20 mM Mops,2 mM MgCl2, 0.1-1 mM EGTA, 3 mM NaN3, 0.5 mMDTT, 0.2-0.5 mM ADP (pH 6.8) (the presence of ADPand 0.5 mM DTT protects HMM from aggregation andinactivation). Trypsin inhibitor was removed by Sepha-dex G-100 gel ®ltration on a 2.6 cm � 90 cm column:2.5 ml fractions were collected with a ¯ow rate of 0.5-1 ml/minute. The fractions containing protein in excessof 1 mg/ml were combined and if needed were concen-trated by ammonium sulfate precipitation. The prep-aration could be frozen rapidly and stored in liquidnitrogen. The sample retained its activity, calcium sensi-tivity, sedimentation characteristics and appearance inthe electron microscope, provided thawing was rapid(achieved by placing the vials in a 20 �C water bath for®ve minutes). The yield was about 10 mg HMM/gmyosin.

Two HMM species were produced by this method,one with a tail 20-23 nm longer than the other. The``short'' and ``long'' HMM preparations were separatedon Sepharose 4B columns (2.6 cm � 90 cm) in 0.6 MNaCl, 20 mM Mops, 2 mM MgCl2, 0.1-1 mM EGTA,3 mM NaN3, 0.5 mM DTT, 0.2 mM ADP (pH 6.8). Initialfractions contained enriched long HMM. To obtainenriched short HMM, chromatography of the later frac-tions had to be repeated.

HMM modifications

The MgADP.Vi complex of HMM was formed asdescribed by Kalabokis & Szent-GyoÈrgyi14 MgADP.A1F4,

and MgADP.BeFx complexes were obtained by incu-bation with 2 mM MgADP, 8 mM NaF, 2 mM BeCl2 orA1(NO3)4 in 80 mM NaC1, 2 mM MgC12, 0.1 mMEGTA, 3 mM NaN3 50 mM Mops, pH 7 on ice for60 minutes. The extent of complex formation was deter-mined from the inhibition of the MgATPase activity,which exceeded 97 %.

Electron microscopy

HMM tail length was measured by electronmicroscopy after samples were diluted to 50 mg/ml in600 mM CH3COONH4 0.5 mM MgADP, 50 % (v/v) gly-cerol (pH 7.0), sprayed onto freshly cleaved mica, andthen rotary shadowed with platinum at a 6 � angle.11 Asimilar procedure was followed to determine the headorientations of cross-linked HMM, in which case the pro-tein at 60 mg/ml concentration was incubated for onehour at 4 �C with 0.3 % (w/v) glutaraldehyde in 80 mMNaCl, 2 mM MgCl2, 0.5 mM ADP, 0.5 mM DTT, 3 mMNaN3, 0.1 mM EGTA, 20 mM Mops (pH 7.0), in the pre-sence or absence of 1 mM CaCl2. The sample was thenmixed with an equal volume of glycerol before sprayingand rotary shadowing. Attempts to perform cross-link-ing with glutaraldehyde at room temperature wereunsuccessful owing to a large amount of HMM aggrega-tion and also polymerization of the glutaraldehyde itself.

A modi®ed mica sandwich technique47 was used toexamine the head orientations of native (unmodi®ed)HMM. A drop of HMM sample diluted to 3 mg/mlusing the above buffer in the absence of glutaraldehydewas adsorbed onto freshly cleaved mica for about60 seconds at room temperature. Excess protein wasrinsed off with sample buffer, then the adsorbed proteinwas ®xed by treatment with 2 % (w/v) uranyl acetate,followed by a ®nal rinse with 100 mM CH3COONH4

containing 30 % glycerol. After the removal of excess¯uid, the sample was rotary shadowed as above.

Grids were examined in either a JEOL 100CX or aPhilips CM10 electron microscope operated at 80 kV.

Analytical ultracentrifugation

Sedimentation velocity experiments were carried outon a Beckman Instruments Optima XL-I Analytical Ultra-centrifuge equipped with a real-time video-based dataacquisition system and Rayleigh optics. The cells wereequipped with sapphire windows and 12 mm alumi-num-®lled epon centerpieces. Sedimentation velocity pat-terns were acquired with the on-line Rayleigh systemevery eight seconds. Apparent sedimentation coef®cientdistribution patterns were computed by the time deriva-tive method using signal averaging48,49 as described byStafford.50 Boundaries were analyzed using the timederivative method.48 Protein concentrations were typi-cally in the range 0.1 to 1.0 mg/ml. All protein solutionswere dialyzed against their respective buffers and thedialysate was used for all dilutions. Runs were per-formed at 20 �C unless otherwise noted. Molecularweights were computed from sedimentation velocitypro®les. Values of s20,w and D20 obtained from the ®ttingprocedure were substituted into the Svedberg equationto obtain the molecular weight.51 Values of the partialspeci®c volume and hydration were computed from the

Changes in Scallop Heavy Meromyosin by Calcium 145

amino acid sequence52 using the consensus partialvolumes,53 and hydration data,54 respectively.

Hydrodynamic modeling

Hydrodynamic modeling was carried out using beadmodeling methods55 with the software programHYDRO.56 The HMM molecule was represented by thebead models shown in Figure 5(a). It consisted of anassemblage of beads of varying sizes. Independentrotation of the heads was allowed in all possible orien-tations in space except for those allowing overlap.A bend in the tail 20 nm from the C terminus of longHMM was assumed to be in a plane. A discrete set ofhead and tail angles (y1, y2, y3) were chosen and thehydrodynamic modeling carried out using HYDRO. Foreach value of y1 chosen for head #1, the values of s20,w

obtained for the various values of y2 chosen for head #2were ®t to a quadratic in y2 to obtain a continuous func-tion of s20,w versus y2. The weight average value of s wascalculated by averaging over all available angles for theheads with sin(y) weighting.57 Averaging over the tailangles was done with equal weighting, since the bendwas restricted to a plane. The bend in the tail resulted inessentially the same increase in s20,w for all angles of theheads and was about 4.6 %. This correction was applieduniformly to all models for all angles of the heads whosehydrodynamic properties were computed for the straighttail con®guration. The results for individual models areshown in Figure 5(b).

Other methods

The relative concentration of the two HMM heavychains was measured by densitometry of SDS/7.5 %polyacrylamide gels58 (E-C Apparatus Co., St. Peters-burg, FL). Protein concentration was determined by Coo-massie blue59 or by biuret tests.60 Mg-ATPase activitywas measured by a coupled assay.61

Acknowledgments

We dedicate this paper to the memory of Michael P.Jacobsen, who died during the completion of this work.We thank Christine Powers for technical assistance withthe shadowing experiments. This work was supportedby NIH grants AR34711 (R.C.), AR15963, AR41803(A.G.S.), NFS grant BIR-9513060 (W.F.S.) and by theCore Electron Microscopy Facility of the University ofMassachusetts Medical School.

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Edited by J. Karn

(Received 31 October 2000; received in revised form 26 January 2001; accepted 29 January 2001)