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Protein Engineering vol. 6 no. 1 pp. 81-84, 1993
© 1993 Oxford University Press

RESEARCH-ARTICLE

Comparative stability of dihydrofolate reductase mutants in vitro and in vivo

Vladimir V. Leontiev1, Vladimir N. Uversky and Anatoly T. Gudkov

Institute of Protein Research, Russian Academy of Sciences 142992 Pushchino, Moscow Region, Russia

1To whom correspondence should be addressed

Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35 {Rightarrow} Asp mutant, Arg57 {Rightarrow} His mutant and the mutant with triple replacement Thr35 {Rightarrow} Asp, Asn37 {Rightarrow} Ser, Arg57 {Rightarrow} 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 {Rightarrow} Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35 {Rightarrow} 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.

Keywords: rigid tertiary structure/protein stability

Received April 14, 1992; revised September 16, 1992; accepted September 30, 1992.


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