Folding Mechanisms of Dihydrofolate Reductase

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MatthewsMCB 0081076The 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 the native conformation. Biophysical and protein engineering techniques will be employed to study a set of homologous proteins that have low sequence similarity but nearly identical three-dimensional structures. Dihydrofolate reductases (DHFR) from Escherichia coli (ecDHFR), Lactobacillus casei (lcDHFR) and human (hDHFR) sources all belong to the alpha/beta sheet class of proteins and contain an embedded nucleoside-binding domain (NBD) that appears to play a critical role in folding. Small angle x-ray scattering and fluorescence resonance energy transfer (FRET) will be used to probe for residual structure in urea-denatured ecDHFR. Mutational analysis will be used to test for the contribution of hydrophobic clusters to the residual structure and to their role in the formation of parallel folding channels previously identified in the ecDHFR folding reaction. Microsecond folding reactions will be monitored by interfacing FRET technology to an ultrafast continuous-flow mixing system. Mutational analysis will be used to test the hypothesis that alternative docking modes between the loop domain and NBD are responsible for the parallel channel folding mechanism. Systematic fluorescence and circular dichroism studies will reveal the folding mechanism of lcDHFR. Comparison with the mechanisms for ecDHFR and hDHFR will test the hypothesis that folding mechanisms of homologous proteins are better conserved than the amino acid sequences. Because the NBD is one of the top ten motifs found in all three super-kingdoms, it is expected that the insights obtained from these studies of the folding mechanisms of a set of DHFRs will be applicable to the folding of a large class of proteins.An understanding of the mechanism by which the sequence of a protein dictates its folding pathway and, ultimately, structure would have a major impact on the prediction of the three-dimensional structure from sequence, on the de novo design of proteins, and on the production of protein products by the biotechnology industry. This research will provide valuable instruction in experimental design, modern biophysical and protein chemistry techniques, and computer-based data analysis to undergraduate students, graduate students and postdoctoral research associates.