Abstract
Transient kinetic measurements of the actomyosin ATPase provided the basis of the Lymn-Taylor model for the cross-bridge cycle, which underpins current models of contraction. Following the determination of the structure of the myosin motor domain, it has been possible to introduce probes at defined sites and resolve the steps in more detail. Probes have been introduced in the Dicytostelium myosin II motor domain via three routes: (i) single tryptophan residues at strategic locations throughout the motor domain; (ii) green fluorescent protein fusions at the N and C termini; and (iii) labelled cysteine residues engineered across the actin-binding cleft. These studies are interpreted with reference to motor domain crystal structures and suggest that the tryptophan (W501) in the relay loop senses the lever arm position, which is controlled by the switch 2 open-to-closed transition at the active site. Actin has little effect on this process per se. A mechanism of product release is proposed in which actin has an indirect effect on the switch 2 and lever arm position to achieve mechanochemical coupling. Switch 1 closing appears to be a key step in the nucleotide-induced actin dissociation, while its opening is required for the subsequent activation of product release. This process has been probed with F239W and F242W substitutions in the switch 1 loop. The E706K mutation in skeletal myosin IIa is associated with a human myopathy. To simulate this disease we investigated the homologous mutation, E683K, in the Dictyostelium myosin motor domain.
Highlights
The proposal of the sliding filament hypothesis in 1954 challenged the prevailing idea that the contractile proteins themselves underwent large-scale conformational changes to cause shortening of muscle (Huxley & Hanson 1954; Huxley & Niedergerke 1954)
The coupling of this mechanical cycle to the ATPase cycle was clarified by transient kinetics studies of the isolated proteins in solution by Lymn & Taylor (1971)
It was shown that ATP binding caused the dissociation of actomyosin before the hydrolysis step and that actin rebound to the myosin–products complex to achieve activation of the ATPase
Summary
The proposal of the sliding filament hypothesis in 1954 challenged the prevailing idea that the contractile proteins themselves underwent large-scale conformational changes to cause shortening of muscle (Huxley & Hanson 1954; Huxley & Niedergerke 1954). The nucleotide-binding isomerizations cannot be resolved from the dissociation phase, and so the influence of actin on the open–closed transition (step 3a) cannot be determined unambiguously (i.e. the ternary AÁMyÁATP and AÁMÃÁATP complexes do not exist in sufficient concentration during their transient formation to determine their ratio) This problem was overcome using ATPcS as the dissociating nucleotide, where studies with the W129þ construct showed that the nucleotide binding events were complete before significant dissociation occurred. ADP binding caused a more limited and much slower reversal of the quench of acto-pyrene-labelled motor domain fluorescence, in line with only partial and slow dissociation These data added weight to the argument that pyrene excimer fluorescence is monitoring a cleft movement that is a requirement for actin dissociation
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