Probing the role of an active site aspartic acid in dihydrofolate reductase

Analogues of E. coli dihydrofolate reductase (DHFR) containing modified amino acids at single, predetermined sites have been prepared. This was accomplished by the use of the DHFR gene containing an engineered nonsense codon (TAG) at the positions corresponding to Val-10 and Asp-27. Misacylated suppressor tRNAs activated with the modified amino acids of interest were employed for the suppression of the nonsense codons in a cell free protein biosynthesizing system, thereby permitting the elaboration of the desired protein analogues. In this fashion, the aspartic acid analogues erythro-carboxyproline, cysteic acid, β,β-dimethylaspartic acid, α-methylaspartic acid, erythro- and threo-β-methylaspartic acid, N-methylaspartic acid, and phosphonoalanine were incorporated into one or both of the aformentioned positions. Although a number of these analogues were incorporated only in low yield, a modification of the strategy has suggested how this might be improved significantly. The derived proteins were purified ...