The adaptability of Escherichia coli thioredoxin to non‐conservative amino acid substitutions

Abstract

The adaptability of Escherichia coli thioredoxin to the substitution of a series of non-natural amino acids has been investigated. Different thiosulfonated alkyl groups were inserted into the hydrophobic core of the protein in position 78 via disulfide bonding with a buried cysteine residue as previously described (Wynn R, Richards F M . 1993. Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. Protein Sci 2:395-403). The side chains added to the cysteine included methyl, ethyl, n-propyl, n-butyl, n-pentyl, and cyclo-pentyl derivatives. The side chains appear to exploit the presence of the large cavities to incorporate these variant forms, enabling the protein to fold and have some activity. Solution structural and kinetic data suggested that these substitutions had little effect on the overall fold of the protein. Thermodynamic 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 dimers despite the limited structural perturbations. Molecular modeling studies allow the conformation of the side chains to be assessed.

Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts

“…E. coli thioredoxin is a particularly difficult molecule to crystallize, and this has inhibited structural characterization of the many mutants of this protein that have been constructed. [40][41][42][43] The activities of these mutant dimers were examined. In addition, a database analysis was carried out to examine the general applicability of this methodology to other proteins.…”

Why we are made of proteins and nucleic acids: Structural biology views on extraterrestrial life

“…Furthermore, the presence of unfilled spaces in such cores would have been advantageous, as they could easily tolerate almost unavoidable contaminations with different amino acids by prebiotic chemistry or primitive translation systems. Such adaptability for random or bulky mutations is even seen in modern proteins [60,61].…”