Equilibrium folding studies of tetrameric R67 dihydrofolate reductase.

Abstract:

:R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded enzyme that confers resistance to the antibacterial drug trimethoprim. This enzyme is not homologous in sequence or structure to chromosomal DHFRs. Equilibrium folding of tetrameric R67 DHFR was studied and found to be fully reversible. Formation of an inactive intermediate was assayed by loss of enzyme activity. Denaturation of the intermediate was monitored by concurrent changes in fluorescence and circular dichroism signals. Both transitions are protein concentration dependent. A simple model fitting these data is tetramer<==>2 dimers<==>4 unfolded monomers. No evidence for folded monomer was found. Global fitting of all the folding data yielded a delta GH2O of -9.63 kcal/mol for the initial transition and a delta GH2O of -12.35 kcal/mol for the second transition. In addition, thermal unfolding of tetrameric R67 DHFR was found to be reversible A folding intermediate also occurred during thermal unfolding as evidenced by the asymmetric endotherms and a delta Hcalorimetric/delta H(van't Hoff) ratio of 2.1.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Zhuang P,Eisenstein E,Howell EE

doi

10.1021/bi00180a018

subject

Has Abstract

pub_date

1994-04-12 00:00:00

pages

4237-44

issue

14

eissn

0006-2960

issn

1520-4995

journal_volume

33

pub_type

杂志文章