The Location and Rate of the Phosphate Release Step in the Muscle Cross-Bridge Cycle.

It is controversial whether the phosphate (Pi) release step in the cross-bridge cycle occurs before or after the first tension-generating step and whether it is fast or slow. We have therefore modified our previous model of the frog cross-bridge cycle by including a Pi release step either before (model A) or after (model B) the first tension-generating step and refined the two models by downhill simplex runs against experimental data for the force-velocity relation and the tension transients after length steps. Pi release step was initially made slow (70 s), but after refinement, it became fast (∼500 s for model A and ∼6000 s for model B).

The force-generation process in active muscle is strain sensitive and endothermic: a temperature-perturbation study.

In experiments on active muscle, we examined the tension decline and its temperature sensitivity at the onset of ramp shortening and at a range of velocities. A segment (∼1.5 mm long) of a skinned muscle fibre isolated from rabbit psoas muscle was held isometrically (sarcomere length ∼2.

Reinterpretation of the Tension Response of Muscle to Stretches and Releases.

We have reexamined the experimental time courses of tension in frog muscle after rapid length steps. The early tension recoveries are biexponential. After 3 nm/hs stretches and releases, the rates of the immediate rapid tension changes are similar but the subsequent tension fall after a stretch is much slower than the rise after a release.

The endothermic ATP hydrolysis and crossbridge attachment steps drive the increase of force with temperature in isometric and shortening muscle.

The isometric tetanic tension of skeletal muscle increases with temperature because attached crossbridge states bearing a relatively low force convert to those bearing a higher force. It was previously proposed that the tension-generating step(s) in the crossbridge cycle was highly endothermic and was therefore itself directly targeted by changes in temperature. However, this did not explain why a rapid rise in temperature (a temperature jump) caused a much slower rate of rise of tension than a rapid length step.

A cross-bridge cycle with two tension-generating steps simulates skeletal muscle mechanics.

We examined whether cross-bridge cycle models with one or two tension-generating steps can account for the force-velocity relation of and tension response to length steps of frog skeletal muscle. Transition-state theory defined the strain dependence of the rate constants. The filament stiffness was non-Hookean.

Crossbridge and filament compliance in muscle: implications for tension generation and lever arm swing.

The stiffness of myosin heads attached to actin is a crucial parameter in determining the kinetics and mechanics of the crossbridge cycle. It has been claimed that the stiffness of myosin heads in the anterior tibialis muscle of the common frog (Rana temporaria) is as high as 3.3 pN/nm, substantially higher than its value in rabbit muscle (~1.

Stabilization of helical order in the thick filaments by blebbistatin: further evidence of coexisting multiple conformations of myosin.

The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation.

Probing muscle myosin motor action: x-ray (m3 and m6) interference measurements report motor domain not lever arm movement.

The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps.

Structural studies of arthrin: monoubiquitinated actin.

Here, we report on the structure and in situ location of arthrin (monoubiquitinated actin). Labelling of insect muscle thin filaments with a ubiquitin antibody reveals that every seventh subunit along the filament long-pitch helices is ubiquitinated. A three-dimensional reconstruction of frozen-hydrated arthrin filaments was produced.

Evaluation of the symmetric model for myosin-linked regulation: effect of site-directed mutations in the regulatory light chain on scallop myosin.

Regulatory myosins are controlled through mechanisms intrinsic to their structures and can alternate between activated and inhibited states. However, the structural difference between these two states is unclear. Scallop (Pecten maximus) striated adductor myosin is activated directly by calcium.

Generalized Crick equations for modeling noncanonical coiled coils.

Crick envisaged the alpha-helical coiled coil to result from systematic bending of an alpha-helix such that every seventh residue was structurally equivalent, and he derived equations for the coordinates of the backbone atoms. Crick's predictions were vindicated experimentally and coiled-coil sequences were shown to have hydrophobic residues alternately spaced 3 and 4 residues apart. Nonetheless, in some coiled coils such canonical heptad repeats are interrupted by inserts of 3 or 4 residues generating decad and hendecad motifs.