Rhodanese mutants containing sequential NH 2 -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 NH 2 -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 ⌬1-10 and ⌬1-11 showed considerable activity. Truncations in the NH 2 terminus affect the chemical stability of the distantly located active site. Residues Ser-11 through Gly-22, which form the NH 2 -proximal ␣-helix, contribute to folding to an active conformation, to resisting degradation during heterologous expression, and to chemical stability in vitro.The enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) catalyzes in vitro the transfer of sulfur from thiosulfate to a number of acceptors including cyanide, lipoic acid, or dithiothreitol (1). The protein is imported into the matrix of the mitochondrion from its site of translation in the cytosol without proteolytic processing of its NH 2 terminus (2). Rhodanese has become an important model for studying issues related to the problems of protein folding (3,4). This monomeric protein is folded into two similar independent, equal-sized domains. The NH 2 -terminal sequence of rhodanese is located primarily on the surface of the NH 2 -terminal domain of the protein (1, 5, 6). Residues Val-1 to Gln-3 appear to be flexible, since they are poorly resolved in the x-ray structure. Residues Val-4 to Arg-7 are fixed to the surface of the NH 2 domain. Residues Ala-8 to Val-10 contribute one of five strands of a -sheet that runs into the interior of the NH 2 domain, and residues Ser-11 to Gly-22 form one of five ␣-helices that surround the -sheet. The residue Lys-23 starts a turn into the interior of the NH 2 domain. All of the residues required for catalysis, including the essential Cys-247, are on the C-terminal domain. In one view then, the C-terminal domain may be active alone. This has never been observed, and it has not been possible to produce stable COOH-terminal domains either proteolytically or recombinantly. Rhodanese offers a unique opportunity to investigate the influe...
The NH 2 -terminal sequence of rhodanese influences many of its properties, ranging from mitochondrial import to folding. Rhodanese truncated by >9 residues is degraded in Escherichia coli. Mutant enzymes with lesser truncations are recoverable and active, but they show altered active site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH 2 -terminal sequence stabilizes the overall structure. We tested aspects of the conformations of these shortened species. Intrinsic and probe fluorescence showed that truncation decreased stability and increased hydrophobic exposure, while near UV CD suggested altered tertiary structure. Under native conditions, truncated rhodanese bound to GroEL and was released and reactivated by adding ATP and GroES, suggesting equilibrium between native and nonnative conformers. Furthermore, GroEL assisted folding of denatured mutants to the same extent as wild type, although at a reduced rate. X-ray crystallography showed that ⌬1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH 2 -terminal residues that contribute to global stability of the native structure in solution do not significantly alter contacts at the atomic level of the crystallized protein. The two-domain structure of rhodanese was not significantly altered by drastically different crystallization conditions or crystal packing suggesting rigidity of the native rhodanese domains and the stabilization of the interdomain interactions by the crystal environment. The results support a model in which loss of interactions near the rhodanese NH 2 terminus does not distort the folded native structure but does facilitate the transition in solution to a molten globule state, which among other things, can interact with molecular chaperones.
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