Truncations at the NH₂ terminus of rhodanese destabilize the enzyme and decrease its heterologous expression

Rhodanese mutants containing sequential NH₂-terminal deletions were constructed to test the distinct contributions of this region of the protein to expression, folding, and stability. The results indicate that the first 11 residues are nonessential for folding to the active conformation, but they are necessary for attaining an active, stable structure when expressed in Escherichia coli. Rhodanese species with up to 9 residues deleted were expressed and purified. Kinetic parameters for the mutants were similar to those of the full-length enzyme. Compared with shorter truncations, mutants missing 7 or 9 residues were (a) increasingly inactivated by urea denaturation, (b) more susceptible to inactivation by dithiothreitol, (c) less able to be reactivated, and (d) less rapidly inactivated by incubation at 37 °C. Immunoprecipitation showed that mutants lacking 10-23 NH₂- terminal amino acids were expressed as inactive species of the expected size but were rapidly eliminated. Cell-free transcription/translation at 37 °C showed mutants deleted through residue 9 were enzymatically active, but they were inactive when deleted further, just as in vivo. However, at 30 °C in vitro, both Δ1-10 and Δ1-11 showed considerable activity. Truncations in the NH₂ terminus affect the chemical stability of the distantly located active site. Residues Ser-11 through Gly-22, which form the NH₂-proximal α- helix, contribute to folding to an active conformation, to resisting degradation during heterologous expression, and to chemical stability in vitro.