title: Comparative stability of dihydrofolate reductase mutants in vitro and in vivo.
abstract: Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis. The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated. In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability. A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.
authors.affiliation: Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region.
authors.forename: V V
authors.initials: VV
authors.lastname: Leontiev
authors.forename: V N
authors.initials: VN
authors.lastname: Uversky
authors.forename: A T
authors.initials: AT
authors.lastname: Gudkov
mesh_terms: D002474:Cell-Free System; D004795:Enzyme Stability / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006358:Hot Temperature; D016296:Mutagenesis; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014295:Trimethoprim / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
chemical_list: D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D014357:Trypsin