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Repriming the Actomyosin Crossbridge Cycle

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Repriming the Actomyosin Crossbridge Cycle Repriming the actomyosin crossbridge cycle Walter Steffen and John Sleep* Randall Centre, King’s College, London SE1 1UL, United Kingdom Edited by Edwin W. Taylor, University of Chicago, Chicago, IL, and approved June 8, 2004 (received for review January 12, 2004) The central features of the mechanical cycle that drives the con- traction of muscle are two translational steps: the working stroke, whereby an attached myosin crossbridge moves relative to the actin filament, and the repriming step, in which the crossbridge returns to its original orientation. Although the mechanism of the first of these is understood in some detail, that of the second has received less attention. Here, we show that repriming occurs after detachment of the crossbridge from the actin, rather than inter- vening between two actomyosin states with ATP bound [Eisen- berg, E. & Greene, L. E. (1980) Annu. Rev. Physiol. 42, 293–309]. To discriminate between these two models we investigated the single-molecule mechanics of the myosin–actin interaction in the presence of ATP analogues such as GTP, for which the hydrolytic step itself limits the actomyosin GTPase rate to a much lower rate than for ATP. The lifetimes of bound states was proportional to 1͞[GTP], indicating that during the bound period myosin was in the actomyosin rigor configuration. Moreover, despite the very low actomyosin GTPase, the rate of actin binding and formation of the rigor state was higher than with ATP; it follows that most inter- Fig. 1. Models of the actomyosin crossbridge cycle. (A) The L-T alignment of actions with actin result in the release of GTP and not of the biochemical states with the crossbridge cycle. Repriming occurs between products, GDP and phosphate. There was no significant movement dissociated states (M⅐T 3 M⅐ D⅐ P). (B) Eisenberg and Greene’s proposal in of the actin during this interaction, so repriming must occur while which the weak states (M⅐T and M⅐D⅐P) bind to actin in the same preforce myosin is dissociated, as in the original Lymn–Taylor scheme conformation. Repriming occurs between attached states (AM⅐T 3 AM⅐T). [Lymn, R. W. & Taylor, E. W. (1971) Biochemistry 10, 4617–4624]. Italics indicate postforce states. he mechanical events responsible for muscle contraction are working stroke, both take place with myosin attached to actin. coupled to the actomyosin ATPase cycle. According to the T However, the kinetic asymmetry accounts for this point: the basic scheme of Lymn and Taylor (L-T) (1) (Fig. 1A), in the ⅐ weakly bound, negative-force-generating AM T90 state very rap- relaxed state (crossbridge 90° to the filament axes) the myosin Ϫ idly dissociates [Ϸ5,000 s 1 (7)], whereas the positive-force- (M) is bound to ADP (D) and phosphate (P). The binding of generating AM⅐D state is more long-lived by 2 orders of mag- actin (A) to M⅐D⅐P leads to the working stroke, accompanied 90 nitude (5). by product release and the formation of the rigor AM45 state. ATP (T) binds to AM to form AM⅐T, from which actin rapidly Our purpose here was to determine whether the L-T scheme dissociates. Hydrolysis then takes place while myosin is dissoci- correctly locates the repriming step, or whether it accords with ated from actin, and the mechanical repriming of the cross- the alternative proposal of Eisenberg and Greene (5). To bridge, which positions it for the start of the next cycle, is distinguish between the two models we needed to establish associated with this step. Further research by Eisenberg and whether or not repriming takes place while myosin is associated collaborators (2) added important details to the scheme. The with actin, that is, in the part of the cycle between AM and ϩ ⅐ concentration of Ca2 regulates the binding to actin of the AM T. With sufficiently good time resolution this question could so-called ‘‘strong binding’’ set of myosin states (M and M⅐D) but be answered by studying interactions with ATP (see Fig. 2 a and Ϸ has little effect on the binding of the ‘‘weak’’ states (M⅐T and b) but at present event detection is currently limited to 10 ms M⅐D⅐P). This finding suggested that these two sets of states bind and the direct approach is not viable because the answer is to actin in different conformations (2), from which it was natural hidden in the gray, temporally unresolved bands (Fig. 2 a and b). to infer that the transition between these conformations might The difference in positions during the free and bound periods is correspond to the working stroke. Moreover, the free energy a measure of the displacement of the AM state with respect to change between M⅐T and M⅐D⅐P was very small (3), and the two the reference free dumbbell position. For this to be a valid complexes bound to actin with similar affinities (4), at least 2 measure of the working stroke, all of the transitions between the orders of magnitude weaker than that of the strong states. The dissociated state (M⅐T 7 M⅐D⅐P) and the rigor state (AM) must actin association constant appeared to be a marker for the be via product dissociation. However, it has been reported (6, 8, inferred two conformational states. In this case, as argued by 9) that during acto-Sl ATPase Ϸ10% of interactions of the Eisenberg and Greene (5), the logical position for the repriming dissociated myosin states (M⅐T 7 M⅐D⅐P) with actin correspond ⅐ step would be the transition from a strongly bound AM T45 to a to ATP release. If this subset of interactions could be identified, ⅐ weakly bound AM T90 state (see Fig. 1B) and not the hydrolysis they would report on the displacement between the reference step as in the original L-T model. This proposal had an attractive symmetry that was further evidenced by the observation that actin is almost as effective in releasing ATP from M⅐Tasin This paper was submitted directly (Track II) to the PNAS office. releasing the products from M⅐D⅐P (6). At first sight the model Abbreviation: L-T, Lymn and Taylor. appears to suffer from the difficulty that the repriming (effec- *To whom correspondence should be addressed. E-mail: [email protected]. tively a reversal of the working stroke), as well as the forward © 2004 by The National Academy of Sciences of the USA 12904–12909 ͉ PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400227101 Downloaded by guest on September 26, 2021 Fig. 3. Use of a rotating microscope slide to move both traps to the left by the same extent. In our standard configuration, using a 1-mm thick slide, the displacement at the stage is Ϸ20 nm per degree of rotation is submillisecond and quite beyond the time resolution of detecting events in the trap. Although the dissociation rates are slower for the analogues [380 sϪ1,ATP␥S (10); 580 sϪ1 at 10°C, GTP (11)], they are still too fast to allow detection of the ternary complex (AM⅐NTP). There are thus two classes of events associated with M⅐NTP binding to actin. The major class is a binding followed by immediate dissociation and goes undetected. The minor class is binding followed by dissociation of NTP and the formation of AM, and it is this class, perhaps only 1% of the total interactions, which we characterize in this article. Exactly equivalent considerations apply when measuring the working stroke in the presence of ATP. Materials and Methods Biochemistry. The biochemical methods have been described (12). Fig. 2. Predictions for interactions based on the L-T and Eisenberg–Greene In outline, cover slips were coated with a suspension of 1.5 ␮m models. (a and b) In the presence of ATP. An arrowhead marks the working silicia beads in 0.05% nitrocellulose followed by BSA. Rabbit stroke and an arrow marks the repriming event in the two models. For the myosin S1 containing a biotinylated regulatory light chain bound purposes of clarity, the duration of the transitions between detached and fairly selectively to this surface. It was applied at a similar rigor states (gray bands) have been exaggerated (expected to be submillisec- Ϸ ␮ ͞ ond). With the present technology, the details within the gray band cannot be concentration ( 1 g ml) for experiments with ATP and for the resolved. (c and d) In the presence of GTP, showing the way in which the analogues. Dumbbells were formed from biotinylated actin held location of the repriming step affects the position of the dumbbell through- between neutravidin-coated beads. out the bound period. BIOPHYSICS Optical Trapping. The optical trap apparatus has been described (12). Because the dumbbell does not rotate about the axis of the position (dissociated state) and AM accessed via ATP release actin filament, to obtain the working stroke it is necessary to and allow assignment of the location of the repriming step. move the dumbbell past the myosin molecule by an integral Ϸ Unfortunately, the amplitude of the brownian noise (SD 10 number of actin repeats (n ϫ 37 nm) past the myosin during the nm) relative to that of the working stroke prevents separation, period of data collection (12). We find that if the traps are moved which results in the interactions observed during ATP hydrolysis by the application of a linear voltage ramp to the acousto-optic Ϸ being a mixture of the product release events of interest ( 90%) deflectors, the resultant motion of the trapped beads can show Ϸ and the contaminant ATP release events ( 10%).
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