A structure—function study of dihydrofolate reductase by protein engineering

Abstract

Several mutants of the enzyme dihydrofolate reductase (DHFR) have been engineered by oligonucleotide-directed mutagenesis of the cloned E. coli gene. The mutations were designed to address specific questions about DHFR structure-function relations that arose from the analysis of the high-resolution structure. Mutations at the active site have revealed that the invariant residue aspartate-27 is involved in substrate protonation, and not in transition-state stabilization as previously thought. The 2.0 Å (1 Å = 10 -1 nm = 10 -10 m) refined structures of the Asn-27 and Ser-27 mutant enzymes reveal that the enhanced binding observed for the 2,4-diamino pteridine and pyrimidine inhibitors is probably not attributable to the charge interaction between Asp-27 and a protonated N-1 of the inhibitor. Substitution of a cysteine for a proline at position 39 places two sulphydryls within bonding distance, and under certain oxidation conditions they will quantitatively form a disulphide bond. The refined 2.0 Å structures of both reduced and oxidized forms of this mutant show that only minor conformational changes occur for disulphide bond formation. The crosslinked enzyme is significantly more conformationally stable to denaturants such as guanidine hydrochloride and urea.

Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding.

“…In some instances genetic screens have allowed the selection of mutant proteins that are more stable than their parent (1)(2)(3). In other cases increased stability has been obtained by rational modifications of the protein structure (4)(5)(6)(7)(8)(9)(10)(11). However, general methods of increasing protein stability are lacking.…”

Dramatic thermostabilization of yeast iso-1-cytochrome c by an asparagine----isoleucine replacement at position 57.

“…However, certain amino acid replacements at specific sites can enhance stability. While general methods for stabilizing proteins are not yet known, genetic selection and screening (1)(2)(3)(4)(5) and rational approaches (6)(7)(8)(9)(10)(11)(12)(13)(14) have resulted in producing mutant proteins with slightly increased stability. We have combined genetic selection with oligonucleotide-directed mutagenesis to produce an altered iso-1-cytochrome c with an unusually large increase in thermal stability.…”

“…Some of the previous approaches used to obtain thermostable proteins are as follows: (i) introduction of disulfide bonds (9,11,13), (ii) exploitation of the difference in configurational backbone entropy between native and denatured states (14,29), and (iii) alteration of specific residues to increase a-helical stability (7,8,30 (14), the amino-terminal domain of the bacteriophage A repressor (8,31), the serine protease subtilisin (5), or the a subunit of tryptophan synthase (32).…”

Single-Molecule Sampling of Dihydrofolate Reductase Shows Kinetic Pauses and an Endosteric Effect Linked to Catalysis

“…The percentage of nonreactive configurations increased with the pH (from 62.6 ± 1.9% at pH 7.15 to 89.0 ± 2.8% at pH 9.1, Table S1 and Figures S14, S17, and S18), while the duration of the nonreactive configurations remained constant over the pH range tested (∼20 ms, Table S1). In DHFR, the protonation of DHF is the rate-limiting step . Since the duration of the reactive configuration is constant over the pH, these data suggest that the protonation of the substrate happens before the M20 loop closes over the reactants, most likely as soon as DHF binds to the enzyme.…”

“…In DHFR, the protonation of DHF is the rate-limiting step. 56 Since the duration of the reactive configuration is constant over the pH, these data suggest that the protonation of the substrate happens before the M20 loop closes over the reactants, most likely as soon as DHF binds to the enzyme.…”