title: Effects of five-tryptophan mutations on structure, stability and function of Escherichia coli dihydrofolate reductase.
abstract: To elucidate the roles of tryptophan residues in the structure, stability, and function of Escherichia coli dihydrofolate reductase (DHFR), its five tryptophan residues were replaced by site-directed mutagenesis with leucine, phenylalanine or valine (W22F, W22L, W30L, W47L, W74F, W74L, W133F, and W133V). Far-ultraviolet circular dichroism (CD) spectra of these mutants reveal that exciton coupling between Trp47 and Trp74 strongly affects the peptide CD of wild-type DHFR, and that Trp133 also contributes appreciably. No additivity was observed in the contributions of individual tryptophan residues to the fluorescence spectrum of wild-type DHFR, Trp74 having a dominant effect. These single-tryptophan mutations induce large changes in the free energy of urea unfolding, which showed values of 1.79-7.14 kcal/mol, compared with the value for wild-type DHFR of 6.08 kcal/mol. Analysis of CD and fluorescence spectra suggests that thermal unfolding involves an intermediate with the native-like secondary structure, the disrupted Trp47-Trp74 exciton coupling, and the solvent-exposed Trp30 and Trp47 side chains. All the mutants except W22L (13%) retain more than 50% of the enzyme activity of wild-type DHFR. These results demonstrate that the five tryptophan residues of DHFR play important roles in its structure and stability but do not crucially affect its enzymatic function.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: E
authors.initials: E
authors.lastname: Ohmae
authors.forename: Y
authors.initials: Y
authors.lastname: Sasaki
authors.forename: K
authors.initials: K
authors.lastname: Gekko
mesh_terms: D002942:Circular Dichroism; D004795:Enzyme Stability / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed / Q000235:genetics* / Q000502:physiology; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism; D014508:Urea / Q000737:chemistry
chemical_list: D014364:Tryptophan; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase