This proposal represents a combined Instrument Development for Biological Research and a Multi-User Biological Equipment and Instrumentation Resources grant application focused on analytical ultracentrifugation. This technique has a wide range of applications: molecular weight analysis, the characterization of molecular shapes and conformational changes, buoyant density separations and the evaluation of association constants from both sedimentation equilibrium and velocity data. Beckman Instruments has developed a new digitally equipped "state of the art" XL-A analytical ultracentrifuge. This instrumentation coupled with the development of very powerful software for data analysis has generated a renaissance in sedimentation analysis. As the research demand for analytical ultracentrifuge measurements increases, and institutions are faced with limited resources for equipment, it becomes very important to consider techniques that can increase the efficiency of instrument usage and approaches that advance the analytical capabilities of sedimentation analysis. The P.I. proposes to pursue the above objectives by using gradient forming cells for the rapid establishment of sedimentation equilbrium thereby increasing instrument efficiency enormously and by developing computational software for the determination of diffusion coefficents using band centrifugation. The following specific aims are proposed for the developmental part of this grant application: 1. The fabrication and testing of a six step double sector gradient forming centerpiece to achieve rapid sedimentation equilibrium. Equilibrium times will be determined for molecular weight sets of spherical proteins; DNA fragments and plasmids. 2. A simultaneous determination od the sedimentation coefficient and diffusion coefficient would provide a rapid evaluation of the molecular weight of a macromolecule. Computational programs will be developed to test the Vinograd and Bruner (1) proposed band centrifugation moment re lationships for the measurement of diffusion coefficents. The band centrifugation analysis will be compared with other sedimentation approaches for obtaining diffusion coefficients. An XL-A analytical ultracentrifuge is essential to accomplish the above specific aims. This instrumentation will also support the research needs of six other investigators at UAB and their research proposals represent the multi-user part of this application. The major users consist of faculty from the Microbiology, Biochemistry, Cell Biology, and the Laboratory of Molecular Biophysics. The Principal Investigator of the grant is Dr. Jacob Lebowitz who has 30 years experience in analytical ultracentrifugation. Most recently the P.I. has spent a month at the National Analytical Ultracentrifuge Facility gaining experience using the NON-LIN program for the analysis of sedimentation equilibrium data for self-associating systems. In addition the P.I. has gained experience in the van Holde-Weischet method for the analysis of boundary sedimentation velocity data. The P.I. is now in an excellent position to rapidly apply the XL-A and data analysis for the research problems of the major users and believes that all the projects proposed in the application can be very significantly assisted by analytical ultracentrifugation. The P.I. believes that the operation of the XL-A and the computational analysis of the data can and should be part of the training of biological scientists working on structure-function problems. The best resolution of research problems rests with those research personnel who will be most directly in need of the results from the sedimentation alalysis. The laboratories of the major users of the XL-A will be required to enroll either a postdoctoral associate or graduate student in a laboratory/lecture course in analytical ultracentrifugation. This training will build a network of investigators that can share knowledge and these individuals can be called upon to assist new users in the operation of the XL-A and in data analysis. After 2 years long term maintenance of the XL-A will be on cost sharing basis provided by the major users. The P.I. believes that the above combination of training and cost sharing will insure the long term use of the XL-A instrumentation. In summary this combined development and multi-user application will provide new developments for sedimentation analysis, assist important research programs and develop in young scientists an expertise in analytical ultracentrifugaton.
