Acquisition of Transient Kinetic Equipment

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Funds are requested to purchase transient kinetic equipment to be employed in research projects from eleven different laboratories at C)region State University. The users represent four departments: Biochemistry and Biophysics, Agricultural Chemistry, the College of Pharmacy and the Department of Food Science and Technology. The equipment will allow the users to pursue new approaches to their respective research problems in the areas of protein folding, protein-protein and protein-DNA interactions, DNA and protein structure, protein- and receptorligand interactions, kinetic studies of enzymes, the coupling of receptors to ion channels and the relationship between mitochondrial integrity, oxidative stress and Ca2+ overload. The availability of this equipment should serve to attract other researchers at Oregon State University interested in mechanistic studies and will therefore serve as an important resource for faculty, postdoctorals, graduate students and undergraduates pursuing these research areas. Since the equipment is novel to Oregon, it may also attract users from other institutions within the state. One major feature of the instrumentation is the ease of data collection and analysis. This should facilitate user training and increase the enthusiasm of users in applying kinetic, mechanistic approaches in their research. The principal investigator is Dr. Michael Schimerlik. The PI received his Ph.D. in enzyme kinetics with Professor W.W. Cleland at Wisconsin and training in transient kinetics while a postdoctoral at The California Institute of Technology with Professor M.A. Raftery where he conducted stopped-flow studies of ligand interactions with the nicotinic acetylcholine receptor. Since coming to Oregon State, the PI has published manuscripts concerning transient kinetic studies in the areas of protein-protein interactions and enzyme mechanisms and is capable of analysis of kinetic mechanisms by fitting data to analytically derived rate equations or, i n more complicated instances, using computer simulations to examine mechanistic models. Several other investigators (Drs. Penner, van Holde, Ho, Anderson and Hsu) also possess expertise in transient kinetic theory. Transient kinetic equipment existing at Oregon State is about fifteen years old, and no longer supported by the manufacturer. No facilities are available for quench flow experiments for use in combination with state of the art facilities at the institution in mass spectrometry or NMR for studies of protein folding. No facilities are available for rapid acquisition of circular dichroism spectra to examine structural changes that occur in protein folding, ligand binding, DNA and protein structural transitions, or macromolecular assembly or dissociation reactions. The abilities to acquire fluorescence or absorbance spectra as a function of time, or to measure time-dependent changes in fluorescence polarization are also lacking. These techniques provide additional sensitivity and enhance the analysis of complicated mechanisms. Most importantly, the software for data acquisition and analysis is far superior to that currently available. In summary, the proposed kinetic equipment will add important new capabilities in transient kinetics not currently available at Oregon State University. In addition it will provide greatly enhanced capabilities over that of our currently available, outdated equipment both in the type of data acquired and in its analysis. This will not only expand the research horizons of laboratories doing kinetic studies but, also because of its "user-friendliness", serve to attract postdoctorals, graduate students and undergraduates so that these individuals will receive training in transient kinetic methods.