Investigation of mutations in a [beta]-bulge region region of dihydrofolate reductase

Author

Amanda Paige Dion

Date of Award

5-1995

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Elizabeth Howell

Committee Members

Jorge Chuchich, Engin Serpersu, Robert Villafane

Abstract

Several second site suppressors of the D27S lesion in Escherichia coli dihydrofolate reductase (DHFR) have been identified. The activity of the primary mutant, D27S DHFR, was found to be greatly decreased at pH 7.0, consistent with aspartic acid-27 being critically involved in proton donation during catalysis. Partial suppressors of the D27S mutation have been genetically selected and identified as F153S or I155N substitutions. D27S+F153S and D27S+I155N DHFRs display 2-3 fold increases in k_cal over D27S DHFR values, but only the F153S mutation decreases K_m, for dihydrofolate by a factor of two. Neither double mutant approaches wild type DHFR activity. Unexpectedly, Phe153 and Ile155 occur on the surface of the protein and are approximately 8 and 14 A distant from the active site. Ile 155 is a member of a β-bulge. A previously identified suppressing mutation, F137S, occurs nearby and is also a member of the same β-bulge [Howell et al., (1990) Biochemistry 29, 8561- 8569]. Clustering of these three second site mutations indicates this area of the structure is important in protein function. Conformational changes due to the presence of these suppressing mutations are likely, as the F153S and I155N mutations do not affect hydride transfer rates upon introduction in wild type DHFR and alterations in circular dichroism spectra are associated with the double mutant DHFRs. To investigate the role of the β-bulge several mutant DHFRs have been constructed in the wild-type and D27S genes. F137 has been deleted to remove the bulge (Δ137 and D27S6+Δ137), and V136 has been replaced with proline, to mimic the right-hand twist imparted by the bulge (V136P+Δ137). The β-blowout from the human DHFR has been inserted into the bulge region (BB and D27S+BB). The kinetics, conformation and stability of these mutant DHFRs have been investigated. Several observations indicate conformational changes occur in these DHFRs including altered circular dichroism spectra and increased k_off rates of NADPH which imply long range effects perturbing the NADPH binding site. The ΔG values of the DHFRs, determined from equilibrium unfolding curves, suggest the mutations also affect the stability of the DHFRs.

Recommended Citation

Dion, Amanda Paige, "Investigation of mutations in a [beta]-bulge region region of dihydrofolate reductase. " PhD diss., University of Tennessee, 1995.
https://trace.tennessee.edu/utk_graddiss/9962