On the origin and transmission of force in actomyosin subfragment 1.

Abstract

A proximity map showing the three-dimensional arrangement of 12 chemically defined points in actomyosin subfragment 1 is developed and roughly correlated with published electron microscope reconstructions of others. Several additional points and topological relationships in the primary polypeptide chain folding are assimilated into this model. Certain crossliikings and distance change observations are interpreted as indicators of transmission of force/displacement between the nucleotide-binding and an actin-binding site-i.e., as indications of how energy is transduced in this system.It is reasonable to think that contractile force arises because the equilibrium geometrical relations between actin and the subfragment 1 (S-1) segment of myosin are different for each enzymatic intermediate occupying an ATPase site of S-1 that is remote from the (S-1)-actin interface(s). Then the operation of the enzymatic cycle compels operation of a cycle of relations between S-1 and actin. If changes in these relations are resisted by (surmountable) external forces, the cycle of relations is a "work cycle" performing external work. In a repetitive system, chemical free energy is thus continuously transduced into mechanical work. One has to say how the binding of an enzymatic intermediate determines an actinbinding relation 5 nm away. We have suggested that binding of the intermediate at the ATPase distorts the local S-1 structure and that this distortion is mechanically transmitted to the distant actin-binding site. These ideas have been developed in previous papers (1-3). This paper fleshes out such ideas with recent observations. Structural Information froin Fluorescence Resonance Energy Transfer (FRET) Mapping and Electron Microscopy (EM)We begin by exploring the structure that may transmit effects between the nucleotide-binding site (N site) and the actinbinding site(s) (A site).A Since crystallography is still unavailable, we resort to melding lesser methods. As shown previously, chemically defined points in acto-(S-1) can be pairwise labeled by fluorophores. From FRET between such a pair one can estimate the interpoint distance, and by repetition of the procedure one can build up a lattice of acto-(S-1) points (2, 5). The improvement offered by the Dale et al. (6) analysis was previously incorporated (2, 7), but since 1984, other developments have appeared (see below). Though we cite improvements we must also emphasize the limitations of proximity mapping inherent in our results. For example, every point lattice has at least one unresolvable "mirror image" ambiguity. More serious is a shortage of necessary distance measurements. Although n(n -1)/2 distances are displayed in a complete lattice of n points, only 4n -10 distances are required to construct the lattice (2). But in our case even this smaller number has not yet been obtained. The missing distances have been assigned by using extraneous information of variable reliability. For example, chemical information may favor the proximity of two points, sequence loc...

Structural interpretation of the mutations in the beta-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy.

“…It is expected that any other residue in this latter location would alter the conformation of this segment and change the domain-domain interactions. Chemical and structural studies of myosin have indicated that there must be a significant structural rearrangement associated with the reactive sulffihydryl groups during the contractile cycle (38)(39)(40)(41). In the structure of chicken skeletal myosin Si, these two residues are separated by an a-helix where their side chains point in opposite directions (23), yet they can be cross-linked in the presence of nucleotide (42)(43)(44) and chemical modification of the cysteines changes the kinetic properties of myosin (45,46).…”

A second consensus sequence of ATP‐requiring proteins resides in the 21‐kDa C‐terminal segment of myosin subfragment 1

“…The proposed assignment of sequence B in myosin patterns represented by EX2RX,GX,(IL)X(FY)X(DE), may have interesting implications regarding the folding Table 2 Results The proximity of elements of the two consensus sequences is also predicted by the recent 3D-lattice model of Botts et al [13], which is based primarily on fluorescence energy transfer measurements. Such an arrangement of the two sequences would imply that regions of both the 27-kDa and 21-kDa segments may contribute to the ATPase site in myosin Sl, and that these two segments, which are well separated in the primary sequence, may not be separate domains as has been suggested from limited proteolysis studies [ 14,151. Recent studies on the progressive unfolding of Sl indicate that the Sl structure may be comprised of a stable domain consisting of the interacting 21-kDa, 27-kDa, and light chain, and a less stable middle 50-kDa segment [ 16,171.…”

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

“…These sites had previously been shown to be remote from each other [94,95]. Because the kinetic data indicate that the binding affinity of S1 distinct ATP and actin-binding sites and in transmitting changes to the lever arm [35].…”