Production and characterization of two single tryptophan mutants of R67 dihydrofolate reductase

One of the fundamental unanswered questions in biochemistry today is; how do proteins fold? R67 dihydrofolate reductase (R67 DHFR) is a well-characterized enzyme that is an excellent model for studying the structural and functional properties of proteins. R67 DHFR is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim (TMP). R67 DHFR is active as a homotetramer with a molecular weight of 34,000. The monomer is 78 amino acids long and has no sequence or structural homology to chromosomal DHFRs. R67 DHFR contains two tryptophan residues per monomer. W45 and W145 as well as the symmetry related residues W245 and W345, occur at the monomer-monomer interfaces. W38 and W338, as well as their symmetry related residues are found at the dimer-dimer interfaces. The positioning of W45 and W38 residues at the monomer and dimer interfaces allow for the utilization of these tryptophans as intrinsic probes to follow the folding/unfolding reactions of R67 DHFR. In these studies, the equilibrium unfolding transitions of R67 DHFR were monitored by following changes in tryptophan fluorescence. Fluorescence spectroscopy is a powerful technique for monitoring conformational changes in proteins. A change in tryptophan environment as monitored by fluorescence generally coincides with a change in the oligomeric state of the protein or a global loss of tertiary and secondary structure, as the unfolding of many proteins are highly cooperative reactions. The dependency of the steady-state fluorescence properties on the concentration of denaturant can provide values for the free energy of folding. The unambiguous assignment of the fluorescent properties to an individual tryptophan residue is necessary for the interpretation of the observed fluorescence signals at a molecular level. There are difficulties however, in resolving the individual contributions of chromophores in multichromophore containing proteins. This limits ones ability to interpret the structural and dynamic properties of these side chains during the unfolding reaction. To over come this limitation the two native tryptophans of R67 DHFR were mutated to the weakly fluorescent amino acid phenylalanine. The goal of this research is to correlate the observed equilibrium unfolding/folding profiles with the individual contributions of the tryptophan residues at each interface. Thus each of the two tryptophans of R67 DHFR were mutagenized to phenylalanine to produce the single Trp mutants, W45F and W38F. The kinetics, oligomeric state, fluorescence and CD-spectral properties of these mutants were then compared to the wild type enzyme.