Bulletin of the American Physical Society
2007 APS March Meeting
Volume 52, Number 1
Monday–Friday, March 5–9, 2007; Denver, Colorado
Session W2: Molecular Motors (Biophysical Society Symposium) |
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Sponsoring Units: DBP Chair: Carlos Catalano, University of Washington Room: Colorado Convention Center Four Seasons 4 |
Thursday, March 8, 2007 2:30PM - 3:06PM |
W2.00001: Pathway of Force Production by the Kinesin-Microtubule ATPase Invited Speaker: Kinesin is the smallest of the molecular motors, consisting of a dimer of motor domains that interact with microtubules and ATP to generate motion towards the plus ends of microtubules for fast axonal transport of membranous organelles. It operates via an alternating site ATPase pathway in which the binding of ATP to one motor domain stimulates the release of ADP from the neighboring domain as the motor walks ``hand over hand'' along the microtubule surface. This alternating site pathway is accomplished in part due to strain that distinguishes the leading from the lagging motor domains when both are bound to the microtubule. This strain leads to a weak nucleotide binding state in the leading motor and a strong nucleotide binding state in the lagging motor. The ATPase activity is linked to alternating weak and strong nucleotide binding states that are coupled to association and dissociation at the microtubule surface to produce a force for forward motion. Strain in the leading motor domain appears to be due to the disruption of the ``neck linker'' in the leading motor. Release of the trailing motor domain from the microtubule surface is the rate-limiting step and, by relaxing the tension, allows the leading domain to bind ATP and continue the cycle and forward motion. Although many of the rate constants for steps in this pathway are known, details regarding the structural and thermodynamic basis for the coupling of ATP hydrolysis to force production remain to be established. I will review our current understanding and describe some of our early attempts to resolve intermediates during movement using single molecule fluorescence methods. \newline \newline In collaboration with Tim Scholz and Bernhard Brenner, Hannover Medical School. [Preview Abstract] |
Thursday, March 8, 2007 3:06PM - 3:42PM |
W2.00002: Invited Speaker: |
Thursday, March 8, 2007 3:42PM - 4:18PM |
W2.00003: Optical tweezers studies of viral DNA packaging: Motor function and DNA confinement in Bacteriophages phi29, lambda, and T4 Invited Speaker: In the assembly of many viruses a powerful molecular motor translocates the genome into a pre-assembled capsid. We use optical tweezers to directly measure translocation of a single DNA molecule into the viral capsid. Improved techniques allow us to measure initiation and early stages of packaging. With phi29 the DNA terminal protein was found to cause large variations in the starting point of packaging. Removal of this protein results in terminal initiation, permitting more accurate assessment of motor function and DNA confinement forces. We investigated the role of electrostatic repulsion by varying ionic screening of the DNA. The observed trends are in accord with those theoretically expected considering counter-ion competition; however the forces are larger than expected in comparison with recent theories and DNA ejection measurements. We have recently succeeded in extending our methods to study two other phages: lambda and T4. These systems have unique structural and functional features, presenting an opportunity for comparative studies in this family of molecular motors. Initial measurements show that lambda and T4 translocate DNA several times faster than the phi29 motor, but are more sensitive to applied load. [Preview Abstract] |
Thursday, March 8, 2007 4:18PM - 4:54PM |
W2.00004: The Viral DNA Packaging Motor of Bacteriophage Lambda Invited Speaker: Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes, which serve as molecular motors that selectively ``package'' viral DNA into a pre-formed procapsid structure, are among the most powerful biological motors characterized to date. Bacteriophage lambda terminase is a heteroligomer composed of gpA and gpNu1 subunits. The smaller gpNu1 subunit is required for specific recognition of viral DNA, a process that is modulated by ATP. The gpA subunit possesses site-specific nuclease and helicase activities that ``mature'' the viral genome prior to packaging. The subunit further possesses a DNA translocase activity that is central to the packaging motor complex. Discrete ATPase sites in gpA modulate the DNA maturation reactions and fuel the DNA packaging reaction. Kinetic characterization of lambda terminase indicates significant interaction between the multiple catalytic sites of the enzyme and has led to a minimal kinetic model describing the assembly of a catalytically-competent packaging motor complex. Biophysical studies demonstrate that purified lambda terminase forms a homogenous, heterotrimeric structure consisting of one gpA subunit in association with two gpNu1 proteins. Four heterotrimers further assemble into a ring-like structure of sufficient size to encircle duplex DNA. The ensemble of data suggests that the ring tetramer represents the biologically relevant, catalytically-competent motor complex responsible for genome processing and packaging reactions. We present a model for the functional DNA packaging motor complex that finds general utility in our global understanding of the enzymology of virus assembly. [Preview Abstract] |
Thursday, March 8, 2007 4:54PM - 5:30PM |
W2.00005: Structural dynamics of myosin V: characterization of the one-head bound intermediate Invited Speaker: Myosin V transports cargo along actin filaments by walking hand over hand. Although this basic model is supported by numerous studies, little is known about the intermediate that occurs when only one of the two heads is bound to actin. Here we use submillisecond darkfield imaging of gold nanoparticle labeled myosin V to directly observe the free head as it releases from the actin filament, diffuses forward, and rebinds. The released head rotates freely about the lever arm junction, a trait which likely facilitates travel through crowded actin meshworks. Free head rebinding occurs more rapidly when one of the six calmodulins bound to the lever arm is replaced with the light chain LC1sa. Our data suggest that strong rebinding and phosphate release occur rapidly, but that the lever arm swing is thwarted by intramolecular strain. The effect of light chain composition on free head rebinding kinetics suggests a potentially elegant means of modulating filament switching and processivity in a tissue-specific manner. [Preview Abstract] |
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