Studies Of Myosin V

Heart, Lung, And Blood Institute

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Abstract

Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have now examined the structure of the inactive complex in more detail using single particle analysis of negatively stained myosin V molecules. Averaged images show considerable detail. The motor domain and lever arm of each molecule bends acutely back upon its stalk and each of the motor domains contact a lobe of the globular tail domain (GTD). The point of contact at the motor domain is a surface loop located near the nucleotide binding site that contains four negatively charged amino acids that are conserved in the myosin V family, but not necessarily in other myosin superfamily members. A truncated HMM-like fragment of myosin V lacking the distal coiled-coil domain of the stalk and the GTD has a high ATPase activity that is not affected by calcium concentration. Addition of a GST-GTD dimeric fusion protein inhibits the ATPase activity of myosin V HMM in the absence, but not the presence of calcium. Examination of the HMM-GST-GTD complex in the absence of calcium shows that the two motor domains of HMM have folded back and contacted the two GTD domains to form a structure remarkably like that seen in the intact molecule. Studies of the frequency of movement of intact myosin V and HMM in the absence of calcium in single molecule motility assays show that the former is markedly inhibited and shows many fewer movements than the latter.[unreadable] [unreadable] We have designed a novel variation of the single-molecule motility assays wherein we use an optical trap to bring beads coated with myosin V to a fimbrin-actin bundle. The trap is released and the run length of the myosin is measured.[unreadable] [unreadable] We have expressed and characterized the kinetic and motile behavior of fragments of human myosin Vc. The detailed kinetic characterization suggests that this motor has a low duty ratio unlike that of the better characterized myosin Va isoforms and is not likely to be processive. Attempts to see processive behavior using single molecule TIRF assays have been unsucessful.[unreadable] [unreadable] A theoretical model of myosin V mechanochemistry is being developed that uses engineering principles to predict the mechanical properties of the lever arm and to model myosin V stepping behavior.