Abstract
We have applied multivariate data analysis to 38.7 nm repeat segments obtained from electron tomograms of isometrically contracting insect flight muscle fibers, mechanically monitored, rapidly frozen, freeze-substituted and thin sectioned. Improved resolution reveals for the first time the helix of F-actin subunits in the thin filament with sufficient clarity that an atomic model can be built into the density independent of the myosin cross-bridges, thereby providing an objective method for identifying weak and strong actin-myosin attachments. The tomogram shows strong binding myosin attachments on only four F-actin subunits midway between successive troponin complexes; these actin subunits comprise the “target zone” of active contraction. Improved quantitation facilitates a more detailed description of weak and strong myosin attachments all along the thin filament including for the first time myosin heads contacting the thin filament on troponin. Most strong binding actin attachments consist of single myosin heads but 28% of bound heads are 2-headed myosin attachments. Strong binding attachments show an axial lever arm range of 77° sweeping out a distance of 12.9 nm. The azimuthal range for the lever arm of strong binding attachments is 127° with a distribution nearly completely to one side of the initial crystallographic structures used for the fitting. There is no apparent coupling between axial angle, representing progress through the power stroke of myosin, and the azimuthal lever arm angle. Two types of weak actin attachments are observed. One type, which is found exclusively on target zone actin subunits, appears to represent prepowerstroke intermediates. The other, which appears to have a different function, is positioned on the M-ward side of the target zone, i.e. the direction toward which filaments slide during sarcomere shortening. Its motor domain contacts tropomyosin rather than actin. Supported by NIH.
Highlights
1125-Plat Three-Dimensional Structure of the Relaxed State of Calcium-Regulated Myosin Filaments John L
We show that whereas wild type (WT) shows a reduction in affinity for exogenously added ADP under loaded conditions, the mutants are relatively insensitive to load
Phosphorylationregulated filaments in the relaxed state are characterized by an asymmetric interaction between the "blocked" and "free" heads of each myosin, inhibiting actin-binding or ATPase activity (Wendt et al, 2001; Woodhead et al, 2005)
Summary
1125-Plat Three-Dimensional Structure of the Relaxed State of Calcium-Regulated Myosin Filaments John L. Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca2þ-binding to the essential light chains, contributing to on-off switching or modulation of contraction. Phosphorylationregulated filaments in the relaxed state are characterized by an asymmetric interaction between the "blocked" and "free" heads of each myosin, inhibiting actin-binding or ATPase activity (Wendt et al, 2001; Woodhead et al, 2005).
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