Effects of multiple replacements at a single position on the folding and stability of dihydrofolate reductase from Escherichia coli.


Abstract

We have made multiple replacements (alanine, arginine, cysteine, histidine, isoleucine, serine, tyrosine) of valine-75 in dihydrofolate reductase from Escherichia coli to examine the relative importance to protein folding of the position that is substituted and the specific character of the amino acid replacement. Valine-75 is part of the eight-stranded beta sheet that forms the structural core of the protein. The isopropyl side chain participates in van der Waals interactions with a number of nonpolar residues, helping to establish a large hydrophobic cluster. Equilibrium studies showed that arginine, histidine, isoleucine, serine, and tyrosine destabilize the protein by 1.9-2.8 kcal mol-1. Alanine and cysteine substitutions have little or no effect. Contrary to other recent studies of the effect of multiple replacements at a hydrophobic site, there is no observed correlation between the changes of the free energy of folding and the changes of the free energy of transfer for the individual amino acids from water to an organic solvent when they are inserted into this site. The effects observed in kinetic studies are both consistent with and extend the equilibrium results; these data indicate that position 75 participates in a rate-limiting step of folding. Some of the equilibrium and kinetic properties of the tyrosine-75 mutant deviated significantly from those of wild-type protein and the other mutants at position 75. (1) The tyrosine variant displayed a complex banding pattern when analyzed by native gel electrophoresis; the wild-type protein and all other mutants at position 75 migrated as single, discrete bands. (2) Comparison of the difference ultraviolet and circular dichroism transition curves showed that a third species is populated at equilibrium; the wild-type protein and all other mutants at position 75 follow a two-state model involving only native and unfolded forms. (3) A third kinetic phase appeared in the unfolding reaction; the wild-type protein and all other mutants at position 75 only showed two kinetic phases in unfolding. Properties 1 and 3 suggest that the tyrosine mutation significantly alters the distribution of native conformers in the protein. These effects on the equilibrium and kinetic data readily display an overriding pattern: residues that would require hydrogen bonding or lead to an expansion of the tightly packed hydrophobic environment in which valine-75 resides destabilize the protein and alter relaxation times of kinetic phases in a consistent manner.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442 Extra Details: additive : K2EDTA(0.2 mM),dG and ddG were measured in the presence of [urea]50%

Submission Details

ID: YtwiW9mY

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:18 p.m.

Version: 1

Publication Details
Garvey EP;Matthews CR,Biochemistry (1989) Effects of multiple replacements at a single position on the folding and stability of dihydrofolate reductase from Escherichia coli. PMID:2655702
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
6CW7 2018-03-30T00:00:00+0000 1.03 E. coli DHFR product complex with (6S)-5,6,7,8-TETRAHYDROFOLATE
6CXK 2018-04-03T00:00:00+0000 1.11 E. coli DHFR substrate complex with Dihydrofolate
6CYV 2018-04-06T00:00:00+0000 1.3 E. coli DHFR ternary complex with NADP and dihydrofolate
1DDR 1995-06-29T00:00:00+0000 2.45 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND UREA
1DDS 1995-06-29T00:00:00+0000 2.2 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE
1DHI 1993-10-29T00:00:00+0000 1.9 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
1DHJ 1993-10-29T00:00:00+0000 1.8 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
1DRA 1991-11-06T00:00:00+0000 1.9 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1DRB 1991-11-06T00:00:00+0000 1.96 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1DRE 1996-11-28T00:00:00+0000 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Dihydrofolate reductase P0ABQ5 DYR_ECOL6
100.0 Dihydrofolate reductase P0ABQ4 DYR_ECOLI
100.0 Dihydrofolate reductase P0ABQ6 DYR_SHIFL
96.2 Dihydrofolate reductase P31073 DYR_CITFR
91.8 Dihydrofolate reductase P31074 DYR_KLEAE