Comparative stability of dihydrofolate reductase mutants in vitro and in vivo

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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.

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