Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese

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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...

Section: Methodsmentioning

confidence: 99%

Truncations at the NH2 Terminus of Rhodanese Destabilize the Enzyme and Decrease Its Heterologous Expression

Trevino¹,

Tsalkova²,

Kramer³

et al. 1998

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“…Rhodanese was prepared as described previously (34) and stored at Ϫ70°C as a crystalline suspension in 1.8 M ammonium sulfate. Rhodanese concentrations were determined using A 280 nm 0.1% ϭ 1.75 (35) and a molecular mass of 33 kDa (36).…”

Section: Methodsmentioning

confidence: 99%

Ligand-induced Conformational Changes in the Apical Domain of the Chaperonin GroEL

Gibbons

Horowitz

1996

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Although the role of nucleotides in the catalytic cycle of the GroESL chaperonin system has been extensively studied, the molecular effects of nucleotides in modulating exposure of sites on GroEL has not been thoroughly investigated. We report here that nucleotides (ATP, ADP, or adenosine 5-(␤,␥-imino)triphosphate) in the presence of Mg 2؉ make the oligomer selectively sensitive to trypsin proteolysis in a fashion suggesting conformational changes in the monomers of one heptameric ring. The site of proteolysis in the monomer that is exposed upon nucleotide binding by the oligomer is in the apical domain (Arg-268). Further, complexes of GroEL with GroES or rhodanese display the same sensitivity to proteolysis, unlike the GroEL-GroES-rhodanese complex, which is protected from proteolysis. The influence of various cations on trypsin proteolysis is investigated to elucidate the differential effects that monovalent and divalent cations have on the oligomeric structure of the chaperonin. These results are discussed in relation to the molecular basis for the chaperonin activity.

“…Proteins-Rhodanese was prepared as described previously (10) and stored at Ϫ70°C as a crystalline suspension in 1.8 M ammonium sulfate. Rhodanese concentrations were determined using A 280 nm 0.1% ϭ 1.75 (11) and a molecular mass of 33 kDa (12).…”

Section: Methodsmentioning

confidence: 99%

Refolding and Reassembly of Active Chaperonin GroEL After Denaturation

Ybarra

Horowitz

1995

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Conditions are reported that, for the first time, permit the folding and assembly of active chaperonin, GroEL, following denaturation in 8 M urea. The folding could be achieved by dilution or dialysis, and the best yields required the simultaneous presence of ammonium sulfate and the Mg 2؉ complexes of ATP or ADP. Ammonium sulfate was the key to this particular protocol, since there was a small recovery of oligomer in its presence, but no detectable recovery was induced by ATP or ADP without ammonium sulfate. The refolded/reassembled GroEL could arrest the spontaneous folding of rhodanese, and it could participate in the chaperonin-assisted refolding of rhodanese as effectively as GroEL that had never been unfolded. The results demonstrate that the primary sequence of GroEL contains the information required for its folding, assembly, and function. Thus, in contrast to previous studies, although chaperonins may facilitate GroEL folding, they are not necessary for the acquisition of the functional oligomeric state of this chaperone. This ability to fold denatured GroEL in vitro will facilitate studies of the influences that determine the interesting folding pattern adopted by the native protein.