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 are not essential for folding or activity. Their replacement with other naturally-occurring amino acids which are not subject to such complications but which maintain essential properties of the protein is a desirable goal. A simple strategy for testing various mutants for their suitability is described for a pair of cysteine residues in dihydrofolate reductase (DHFR) from Escherichia coli. Using a reconstructed gene which preserves the amino acid sequence and introduces a variety of unique restriction sites, the cysteines at positions 85 and 152 were replaced 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 to wild type DHFR. The Cys85-->Ala and Cys152-->Ser double mutant DHFR was found to retain the four channel folding mechanism and have activity and stability which are comparable to the wild type enzyme. The replacement of the cysteines improved the resistance of DHFR to the irreversible loss of activity at high temperature.
Iwakura
M
M
Department of Chemistry, Center for Biomolecular Structure and Function, and Biotechnology Institute, Pennsylvania State University 16802, USA.
Jones
B E
BE
Luo
J
J
Matthews
C R
CR
D000595:Amino Acid Sequence; D001483:Base Sequence; D003553:Cystine / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*
D003553:Cystine; D013762:Tetrahydrofolate Dehydrogenase