Repriming the actomyosin crossbridge cycle

Abstract

The central features of the mechanical cycle that drives the contraction 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 intervening between two actomyosin states with ATP bound [Eisenberg, 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 interactions with actin result in the release of GTP and not of the products, GDP and phosphate. There was no significant movement of the actin during this interaction, so repriming must occur while myosin is dissociated, as in the original Lymn- Taylor T he mechanical events responsible for muscle contraction are coupled to the actomyosin ATPase cycle. According to the basic scheme of Lymn and Taylor (L-T) (1) (Fig. 1A), in the relaxed state (crossbridge 90°to the filament axes) the myosin (M) is bound to ADP (D) and phosphate (P). The binding of actin (A) to M⅐D⅐P 90 leads to the working stroke, accompanied by product release and the formation of the rigor AM 45 state. ATP (T) binds to AM to form AM⅐T, from which actin rapidly dissociates. Hydrolysis then takes place while myosin is dissociated from actin, and the mechanical repriming of the crossbridge, which positions it for the start of the next cycle, is associated with this step. Further research by Eisenberg and collaborators (2) added important details to the scheme. The concentration of Ca 2ϩ regulates the binding to actin of the so-called ''strong binding'' set of myosin states (M and M⅐D) but has little effect on the binding of the ''weak'' states (M⅐T and M⅐D⅐P). This finding suggested that these two sets of states bind to actin in different conformations (2), from which it was natural to infer that the transition between these conformations might correspond to the working stroke. Moreover, the free energy change between M⅐T and M⅐D⅐P was very small (3), and the two complexes bound to actin with similar affinities (4), at least 2 orders of magnitude weaker than that of the strong states. The actin association constant appeared to be a marker for the inferred two conformational states. In this case, as argued by Eisenberg and Greene (5), the logical...

Maximal Activation of Skeletal Muscle Thin Filaments Requires Both Rigor Myosin S1 and Calcium

“…2 However, these myosin-ligand complexes promote an open switch II conformation of myosin (compared with the closed switch II conformation of M-ADP-P i ) (7)(8)(9) and may also change the mechanism of myosin binding to actin. Supporting this view are single molecule mechanical measurements which show that fast skeletal myosin complexed with ligands that produce an open switch II conformation do not produce a power stroke upon binding to actin (10). It is, therefore, likely that the regulation of the binding of myosin to thin filaments in the open conformation may not give an accurate description of the mechanism of the regulation of thin filament accelerated product dissociation from M-ADP-P i .…”

“…It is now generally accepted that myosin exists in two or more conformations and that the conformation is dependent upon the ligands bound to the active site. Of the ligands listed above, only ADP-P i predominately produces the "closed" switch II conformation of the myosin nucleotide binding site that is required to produce a power stroke when the myosin binds to actin (10).…”

Phosphorylation of Cardiac Troponin I at Protein Kinase C Site Threonine 144 Depresses Cooperative Activation of Thin Filaments

“…Statistical Analysis-To determine the pCa value at halfmaximal fluorescence change (pCa 50 ) in reconstituted thin filament or force generation in skinned fiber and the Hill coefficient (n H ), the fluorescence and force were normalized to maximum. The relationship of fluorescence change or force generation with pCa was fit to a modified Hill equation (25,30).…”

Using optical tweezers to relate the chemical and mechanical cross-bridge cycles

“…The rate of actin activated hydrolysis of GTP is some 40 times less than for ATP (10 C, low ionic strength; White et al 1993). We observed that in the optical trap the interaction rate, far from being much lower, was in fact significantly higher with GTP, ITP and ATPcS than with ATP (Steffen & Sleep 2004; see table 2). For dumbbell experiments, the duration of events is strictly controlled by the second-order rate of NTP binding but the rate of observing events is critically dependent on the exact position of the dumbbell with respect to the fixed myosin bearing bead, thus a relatively large number of experiments are needed to make a comparison.…”

“…The displacements of the dumbbell observed in the presence of NTP are shown in table 2 (Steffen & Sleep 2004). In each case, the value is not significantly different from zero and thus we conclude that M Á GTP, M Á ITP and M Á ATPcS all bind to actin in the same conformational state as the final AM rigor state.…”