Masters Theses

Date of Award

5-1998

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Elizabeth Ehrhardt Howell

Committee Members

Daniel Roberts, Cynthia Peterson

Abstract

R67 dihydrofolate reductase (DHFR) is a novel protein that confers clinical resistance to trimethoprim (TMP). Surprisingly, this R-plasmid encoded enzyme does not share any homology with chromosomal DHFR. The crystal structure of active homotetrameric R67 DHFR indicates that a single active site pore transverses the length of the molecule (Narayana et al., 1995). Since the center of the pore possesses exact 222 symmetry, site directed mutagenesis of the gene sequence encoding R67 DHFR will introduce four symmetrically related mutations at the single active site. To break this symmetry, a gene oligomerization strategy has been designed to construct an R67 DHFR hybrid possessing the essential tertiary structure of native tetrameric R67 DHFR. The procedures used in creation of the R67 DHFR hybrid require triple R67 DHFR (a gene triplication product) and a single gene copy containing an N-terrninal Histag sequence (Histag R67 DHFR). The Histag sequence is necessary for purification of the hybrid species utilizing nickel chelating affinity chromatography. Triple R67 DHFR is a covalently linked three domain monomer created by linking in frame three copies of the R67 DHFR gene. The results in this thesis show that both triple and Histag R67 DHFRs are kinetically similar to native R67 DHFR and are accordingly suitable for constructing the hybrid. Two important requirements for creating a functional hybrid from the above gene oligomerization strategy are that both triple and Histag R67 DHFR associate to form a stable structure that kinetically mimics the wildtype homotetramer, and that the N-terrninal Histag sequence does not block access to the active site. The pH titration, gel filtration, and CD data, in this thesis, show substantial evidence that both proteins associate to produce a homogenous hybrid species corresponding to a 1:1 molar ratio of triple and Histag R67 DHFRs. Furthermore, this species is also stable and kinetically similar to the wildtype enzyme. The data shown in this thesis are significant in that they show that the R67 DHFR hybrid is very similar to the native homotetramer. Thus, the next step would be to use this procedure to create a mutant R67 DHFR hybrid to study asymmetric mutations in the active site of R67 DHFR.

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