Folding Mechanisms of Dihydrofolate Reductase and the Response Regulators

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The overall goal of this project is to understand the mechanism by which the amino acid sequence of a protein directs its rapid and efficient folding to a native, functional form. The target of these studies will be several representatives of the alpha/beta/alpha class of protein structures, one of the most common motifs found in biology. Prior work from this laboratory on two members of this class, dihydrofolate reductase (DHFR) and H-ras p21, has revealed complex kinetic processes that begin in the sub-millisecond time range and proceed through transient intermediates in parallel pathways. Future studies will focus on DHFR and several representatives of the flavodoxin fold, including the response regulators CheY, NtrC, and SpoOF. Conventional small angle x-ray scattering instrumentation and a novel time-resolved fluorescence spectroscopy technique developed over the previous grant period will be used to detect non-random structure in unfolded states and monitor folding reactions in the microsecond time range for DHFR, CheY, NtrC, and SpoOF. Genetic engineering methods will be used to create mutant proteins suitable for labeling with fluorophores required for distance measurements using Forster resonance energy transfer techniques. Mutations will also be employed to test the involvement of specific beta strands and alpha helices in non-random structure in the unfolded protein and in guiding the early stages of folding. The information obtained on transient intermediates and the barriers separating them from stable thermodynamics states will enhance the understanding of the folding mechanism of the alpha/beta/alpha fold and provide parameters useful to theorists who simulate folding reactions.The broader impact of this effort is multi-fold. First, support of this research project will enhance the development of a graduate program in Chemical Biology at the University of Massachusetts Medical School. The concepts and methods employed to design experiments, analyze the data, and interpret the results have motivated the creation of a course in Molecular Biophysics which will be taught to first and second year students in the Graduate School for Biomedical Sciences. This course is also available to undergraduates in the Worcester area, including those at Clark University and Worcester Polytechnic Institute. Second, undergraduates, graduate students, and postdoctoral fellows will receive training in molecular biophysics which will serve to advance important research goals and to prepare them for professional careers in industry, academia, and government. Third, novel technology developed during the previous grant period will be refined and applied to the protein folding problem, one of the most significant impediments to realizing the full benefit of the genomic sequencing efforts. A patent disclosure has been filed to encourage the application of this micro-fluidics mixing technology to other areas of biology or chemistry where microsecond reactions occur.