melting's Spudich:JA [11 articles]

Abstract

Myosins have diverse mechanical properties reflecting a range of cellular roles. A major challenge is to understand the structural basis for generating novel functions from a common motor core. Myosin VI (M6) is specialized for processive motion toward the (-) end of actin filaments. We have used engineered M6 motors to test and refine the "redirected power stroke" model for (-) end directionality and to explore poorly understood structural requirements for processive stepping. Guided by crystal structures and molecular modeling, we ...

Abstract

Myosin VI is a molecular motor that can walk processively on actin filaments with a 36-nm step size. The walking mechanism of myosin VI is controversial because it takes very large steps without an apparent lever arm of required length. Therefore, myosin VI is argued to be the first exception to the widely established lever arm theory. It is therefore critical to directly demonstrate whether this motor walks hand-over-hand along actin despite its short lever arm. Here, we follow the displacement ...

Abstract

Observation of discrete, single-molecule binding events allows one to bypass assumptions required to infer single-molecule properties from studies of ensembles of molecules. Optically trapped beads and glass microneedles have been applied to detect single-molecule binding events, but it remains difficult to identify signs of binding events given the large displacements induced by thermal forces. Here, we exploit thermal diffusion by using correlation between motion of optically trapped beads attached to both ends of a single actin filament to track binding events ...

Abstract

10.1073/pnas.0409487102 Here we present a technique called single-molecule high-resolution colocalization (SHREC) of fluorescent dyes that allows the measurement of interfluorophore distances in macromolecules and macromolecular complexes with better than 10-nm resolution. By using two chromatically differing fluorescent molecules as probes, we are able to circumvent the Rayleigh criterion and measure distances much smaller than 250 nm. The probes are imaged separately and localized individually with high precision. The registration between the two imaging channels is measured by using fiduciary markers, and ...

Abstract

Myosin V is an efficient processive molecular motor. Recent experiments have shown how the structure and kinetics of myosin V are specialized to produce a highly processive motor capable of taking multiple 36-nm steps on an actin filament track. Here, we examine how two identical heads coordinate their activity to produce efficient hand-over-hand stepping. We have used a modified laser-trap microscope to apply a approximately 2-pN forward or backward force on a single-headed myosin V molecule, hypothesized to simulate forces experienced ...

Abstract

Myosin VI supports movement toward the (-) end of actin filaments, despite sharing extensive sequence and structural homology with (+)-end-directed myosins. A class-specific stretch of amino acids inserted between the converter domain and the lever arm was proposed to provide the structural basis of directionality reversal. Indeed, the unique insert mediates a 120 degrees redirection of the lever arm in a crystal structure of the presumed poststroke conformation of myosin VI [Ménétrey J, Bahloul A, Wells AL, Yengo CM, Morris CA, ...

Abstract

Dwell-time distributions, waiting-time distributions, and distributions of pause durations are widely reported for molecular motors based on single-molecule biophysical experiments. These distributions provide important information concerning the functional mechanisms of enzymes and their underlying kinetic and mechanical processes. We have extended the absorbing boundary method to simulate dwell-time distributions of complex kinetic schemes, which include cyclic, branching, and reverse transitions typically observed in molecular motors. This extended absorbing boundary method allows global fitting of dwell-time distributions for enzymes subject to different ...

Abstract

A recent study has revealed an unexpected change in conformation of the myosin VI converter domain, essential for twisting the lever arm through a approximately 180 degrees rotation to achieve a large step along actin. ...