COMPARATIVE STABILITY OF DIHYDROFOLATE-REDUCTASE MUTANTS INVITRO AND INVIVO

Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35 double-line arrow pointing right Asp mutant, Arg57 double-line arrow pointing right His mutant and the mutant with triple replacement Thr35 double-line arrow pointing right Asp, Asn37 double-line arrow pointing right Ser, Arg57 double-line arrow pointing right His) were obtained by site-directed mutagenesis. rhe 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 double-line arrow pointing right Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35 double-line pointing right 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.