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

Abstract

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...

Model for kinetics of myosin-V molecular motors

“…Furthermore, according to previous experimental results, the relative orientation of the neck domain with respect to the motor domain in the presence of actin is also dependent on its nucleotide state: In weak actin-bound state (ADP.Pi) the relative orientation is random [24][25][26][27][28][29][30], as schematically shown in Fig. 1 (a).…”

Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle

“…This conformational change may cause distortion of the transducer region (␤-sheet), generating straindependent tension that subsequently results in actin-activated phosphate release through the ''back door'' (33). A strongly bound AM⅐ADP⅐P i state has been proposed based on muscle fiber studies (14), single-molecule laser trap studies (32), and rapid freezing EM studies (34). The large temperature dependence of the conformational change (Q 10 ϭ 3.2) is consistent with a significant domain motion such as cleft closure that proceeds through a large activation energy barrier (U a Ϸ34 k B T).…”

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

“…Thus, only muscle S1s having NTEs were capable of displacing the peptide from actin C-terminal region. On the other hand, FRET was not affected in the presence of ATP, where S1 binds weakly and is rotationally disordered, and the distance between A1NTE and actin significantly increases compared with that in the strongly bound complex (25,26). FRET of actin-ANT complex was also significantly decreased by an increase in ionic strength (0.1 M KCl), further supporting the similarity between ANT-actin and actin-A1NTE interaction; a structural model of the acto-S1A1 complex (19) shows that the positively charged N terminus of A1 binds to a cluster of negatively charged residues in the C-terminal region of actin (19).…”