THE ADAPTABILITY OF ESCHERICHIA-COLI THIOREDOXIN TO NONCONSERVATIVE AMINO-ACID SUBSTITUTIONS

The adaptability of Escherichia coli thioredoxin to the substitution o f a series of non-natural amino acids has been investigated. different thiosulfonated alkyl groups were inserted into the hydrophobic core o f the protein in position 78 via disulfide bonding with a buried cyste ine residue as previously described (Wynn R, Richards FM. 1993. Unnatu ral amino acid packing mutants of Escherichia coli thioredoxin produce d by combined mutagenesis/chemical modification techniques. Protein Sc i 2:395-403). The side chains added to the cysteine included methyl, e thyl, n-propyl, n-butyl, n-pentyl, and cyclo-pentyl derivatives. The s ide chains appear to exploit the presence of the large cavities to inc orporate these variant forms, enabling the protein to fold and have so me activity. Solution structural and kinetic data suggested that these substitutions had little effect on the overall fold of the protein. T hermodynamic data revealed that the entropic effect of restricting the side chains in the folded protein has an effect on the stability. The variant forms of thioredoxin have different propensities to form dime rs despite the limited structural perturbations. Molecular modeling st udies allow the conformation of the side chains to be assessed.