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. Study holds ProTherm entries: 4429, 4430, 4431 Extra Details: tetrameric R67 DHFR; enzyme activity; folding intermediate;,asymmetric endotherms

Submission Details

ID: uQVVzmL5

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Zhuang P;Eisenstein E;Howell EE,Biochemistry (1994) Equilibrium folding studies of tetrameric R67 dihydrofolate reductase. PMID:8155640
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1VIE 1996-10-03T00:00:00+0000 1.7 STRUCTURE OF DIHYDROFOLATE REDUCTASE
1VIF 1996-10-03T00:00:00+0000 1.8 STRUCTURE OF DIHYDROFOLATE REDUCTASE
2GQV 2006-04-22T00:00:00+0000 1.1 High-resolution structure of a plasmid-encoded dihydrofolate reductase: pentagonal network of water molecules in the D2-symmetric active site
2P4T 2007-03-13T00:00:00+0000 1.15 Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode
2RH2 2007-10-05T00:00:00+0000 0.96 High Resolution DHFR R-67
2RK1 2007-10-16T00:00:00+0000 1.26 DHFR R67 Complexed with NADP and dihydrofolate
2RK2 2007-10-16T00:00:00+0000 1.9 DHFR R-67 complexed with NADP
3SFM 2011-06-13T00:00:00+0000 1.4 Novel crystallization conditions for tandem variant R67 DHFR yields wild-type crystal structure
6NXZ 2019-02-10T00:00:00+0000 1.75 Crystal structure of trimethoprim-resistant type II dihydrofolate reductase in complex with a bisbenzimidazole inhibitor
6NY0 2019-02-10T00:00:00+0000 1.4 Crystal structure of trimethoprim-resistant type II dihydrofolate reductase in complex with a bisbenzimidazole inhibitor

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Dihydrofolate reductase type 2 P00383 DYR21_ECOLX