A STRATEGY FOR TESTING THE SUITABILITY OF CYSTEINE REPLACEMENTS IN DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI

Amino acid sequences in proteins can contain residues which complicate biochemical, biophysical, or protein engineering studies but which ar e not essential for folding or activity, Their replacement with other naturally-occurring amino acids which are not subject to such complica tions but which maintain essential properties of the protein is a desi rable goal. A simple strategy for testing various mutants for their su itability is described for a pair of cysteine residues in dihydrofolat e reductase (DHFR) from Escherichia coli. Using a reconstructed gene w hich preserves the amino acid sequence and introduces a variety of uni que restriction sites, the cysteines at positions 85 and 152 were repl aced by site-directed and cassette mutagenesis. The enzymatic activity , stability, and folding mechanism of six double mutant DHFR proteins were examined with the purpose of identifying a suitable alternative t o wild type DHFR. The Cys85 --> Ala and Cys152 --> Ser double mutant D HFR was found to retain the four channel folding mechanism and have ac tivity and stability which are comparable to the wild type enzyme, The replacement of the cysteines improved the resistance of DHFR to the i rreversible loss of activity at high temperature.