This project involves the acquisition and installation of a state-of-the-art-analytical ultracentrifuge in the Department of Molecular Physiology and Biophysics at the University of Vermont. This instrument represents newly developed technology, fully computerized to provide convenient and rapid data acquisition and manipulation. Computer programs are available to perform the many of the computations needed to determine molecular weights, sedimentation and diffusion coefficients and the degree of homogeneity or polydispersity of macromolecules. One of the most important applications of the new technology will be the ability to quantitatively analyze the association-dissociation equilibria of complex macromolecular assemblies that carry out most cellular functions. With the rapid advances in molecular biology and the unprecedented ability to create unlimited new proteins in bacterial and baculovirus expression systems, there is a compelling need to characterize wild type and mutant proteins by precise physical chemical techniques. The analytical ultracentrifuge remains the tool of choice for such studies. The research of the major users is dedicated to understanding the structure and function of macromolecules and their assemblies, in particular, those involved in muscle contraction and non-muscle motility. Most of the projects will use the instrumentation to study some aspect of the molecular motor, myosin, and its interactions with actin; the role of light chain isoforms in cardiac myosins; the regulation of non-muscle myosins by calmodulin, and assembly properties of smooth muscle myosin; forces and motions generated by single myosin molecules; molecular interactions at the actomyosin interface; role of Drosophila myosin regulatory light chain in oscillatory power generation; thin filament regulation in heart failure. Several of the projects will use the instrumentation to characterize the expressed proteins for homogeneity, a property essential for single molecule mechanics in optical traps; and for x-ray crystallography. In addition, this instrument will serve as an excellent teaching tool for graduate students and post-doctoral fellows as well as for undergraduates. Few instruments demonstrate as vividly as this the effects of environmental conditions on the size, shape and interaction properties of biological macromolecules.
