Orientational disorder and motion of weakly attached cross-bridges

Fajer, Piotr G.; Fajer, E. A.; Schoenberg, Mark J.; Thomas, David D.

doi:10.1016/s0006-3495(91)82093-8

Cited by 40 publications

(36 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There are experimental data obtained by several different methods indicating that a transition from disordered to ordered binding of the myosin head while attached to actin produces the power stroke. ESR studies of AM-S1 in solution, myofibrils, and fibers all indicate that a large fraction of myosin heads in contracting muscle fibers are weakly bound to actin and are disordered and highly mobile on the microsecond time scale (26)(27)(28). X-ray diffraction and mechanical studies support this view (29,30).…”

Section: Discussionmentioning

confidence: 96%

Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry

Walker

Zhang

Jiang

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The mechanism of binding of myosin subfragment-1 (S1) to actin in the absence of nucleotides was studied by a combination of stopped-f low f luorescence and ms time resolution electron microscopy. The f luorescence data were obtained by using pyrene-labeled actin and exhibit a lag phase. This demonstrates the presence of a transient intermediate after the collision complex and before the formation of the stable ''rigor'' complex. The transient intermediate predominates 2-15 ms after mixing, whereas the rigor complex predominates at time >50 ms. Electron microscopy of acto-S1 frozen 10 ms after mixing revealed disordered binding. Acto-S1 frozen at 50 ms or longer showed the ''arrowhead'' appearance characteristic of rigor. The most likely explanation of the disorder of the transient intermediate is that the binding is through one or more f lexible loops on the surfaces of the proteins. The transition from disordered to ordered binding is likely to be part of the force-generating step in muscle.The primary force-producing event of muscle contraction is thought to be a gross conformational change in the heads of the myosin (M) molecule whilst attached to actin (A) (1, 2); however, the structural and biochemical steps in this mechanism are not well understood. The tightly bound actomyosin (AM) complex that forms in the absence of nucleotides and probably corresponds to the end of the power-stroke has been described to Ϸ3-nm resolution (3). In smooth muscle, another tightly bound AM complex in ADP has been shown to have a substantially different conformation (4). However, the conformation(s) of the earlier stages in the power-stroke are not known. For instance, it is not known whether the start of the power-stroke has only one conformation, nor is it known whether the various transient intermediates that can be identified by solution kinetics are associated with particular conformations.The biochemistry of the interaction between actin and myosin subfragments has been studied in vitro by a variety of methods, including binding, stopped-flow, and pressure relaxation (5-7). Intermediates of the ATP hydrolysis mechanism have been classified into two broad categories according to actin affinity, weak and strong (8, 9), and the force-producing step is thought to involve conversion from weak to strong binding. Thus myosin subfragment-1 (S1) binds strongly to actin in the absence of nucleotide or with ADP in the active site, but the binding is approximately 10 4 weaker with either ATP or ADP and P i present. Geeves and coworkers have shown that S1 and S1-ADP bind to actin by a two-step mechanism and proposed that the initial transient state is similar to the weak state formed when either ATP or ADP and P i are in the active site. The weak and rigor states have often been referred to as the A and R states, respectively (10).The conformations of the weakly bound complexes have been little studied, in large part because they tend to dissociate at the low protein concentration feasible for electron microscopy of solutio...

show abstract

Section: Discussionmentioning

confidence: 96%

Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry

Walker

Zhang

Jiang

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This leads to an equal distribution between the M1 and M2 LC domain orientations. The catalytic domain is disordered (10,(47)(48)(49)(50)(51). Upon activation, one head of the dimer binds actin without an LC domain rotation.…”

Section: Discussionmentioning

confidence: 99%

Myosin Light-Chain Domain Rotates upon Muscle Activation but Not ATP Hydrolysis

et al. 1999

View full text Add to dashboard Cite

We have studied the correlation between myosin structure, myosin biochemistry, and muscle force. Two distinct orientations of the myosin light-chain domain were previously resolved using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled regulatory light chains in scallop muscle fibers. In the present study, we measured isometric force during EPR spectral acquisition, in order to define how these two light-chain domain orientations are coupled to force and the myosin ATPase cycle. When muscle fibers are partially activated with increasing amounts of calcium, the distribution between the two light-chain domain orientations shifts toward the one associated with strong actin binding. This shift in distribution is linearly related to the increase in force, suggesting that rotation of the light-chain domain is coupled to strong actin binding. However, when nucleotide analogues are used to trap myosin in the pre- and posthydrolysis states of its ATPase cycle in relaxed muscle, there is no change in the distribution between light-chain domain orientations, showing that the rotation of the light-chain domain is not directly coupled to the ATP hydrolysis step. Instead, it is likely that in relaxed muscle the myosin thick filament stabilizes two light-chain domain orientations that are independent of the nucleotide analogue bound at the active site. We conclude that a large and distinct rotation of the light-chain domain of myosin is responsible for force generation and is coupled to strong actin binding but is not coupled to a specific step in the myosin ATPase reaction.

show abstract

“…Because most models of muscle contraction consider the myosin head as the active component in force generation, EPR studies have targeted the rotational motions and orientational distributions of the myosin head (e.g., Thomas and Cooke, 1980;Cooke et al, 1982; Address correspondence to Dr. D. D. Berger et al, 1989;Barnett and Thomas, 1989;Fajer et al, 1991). However, a number of investigations suggest that actin undergoes conformational changes upon the binding of S1 and that these changes are necessary for the activation ofmuscle contraction (e.g., Prochniewicz-Nakayama and Yanagida, 1982;Miki et al, 1987;Prochniewicz and Yanagida, 1990).…”

Section: Introductionmentioning

confidence: 99%

Orientational distribution of spin-labeled actin oriented by flow

Ostap¹,

Yanagida²,

Thomas³

1992

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

Previous studies on spin-labeled F-actin (MSL-actin), using saturation transfer electron paramagnetic resonance (ST-EPR), have demonstrated that actin has submillisecond rotational flexibility and that this flexibility is affected by the binding of myosin and its subfragments. This rotational flexibility does not change during the active interaction of myosin heads, actin, and adenosine triphosphate. However, these ST-EPR studies, performed on randomly oriented actin, would not be sensitive to orientational changes on the millisecond time scale or slower. In the present study, we have clarified these results by performing conventional EPR experiments on MSL-actin oriented by flow to detect changes in the orientational distribution. We have determined the orientational distribution of the spin labels relative to the magnetic field (flow direction) by comparing experimental EPR spectra to simulated EPR spectra corresponding to known orientational distributions. Spectra acquired during flow indicate two populations of probes: a highly ordered population and a disordered population. For the ordered population (28% of the total spin concentration), the angle between the actin filament axis and the nitroxide z axis (theta) fits a Gaussian distribution centered at 32.0 +/- 0.9 degrees, with a full width at half maximum of 20.7 +/- 3.9 degrees. The angle between the nitroxide x axis and the projection of the field in the xy plane (phi) is centered at 37.5 +/- 9.2 degrees with a full width of 24.9 +/- 10.7 degrees. This orientational distribution is not significantly changed upon the binding of phalloidin or myosin subfragment 1 (S1), indicating that these proteins do not affect the axial orientation of actin subunits. Spectra of spin-labeled S1 (MSL-S1) bound to actin oriented by flow have about the same orientational distribution as MSL-S1 bound to actin in oriented fibers. Thus, the oriented fraction of flow-oriented actin filaments has nearly the same high degree of alignment as the actin filaments in muscle fibers.

show abstract

Orientational disorder and motion of weakly attached cross-bridges

Cited by 40 publications

References 36 publications

Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry

Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry

Myosin Light-Chain Domain Rotates upon Muscle Activation but Not ATP Hydrolysis

Orientational distribution of spin-labeled actin oriented by flow

Contact Info

Product

Resources

About