NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions

Richard J. Trevino, Francesca Gliubich, Rodolfo Berni, Michele Cianci, John Chirgwin, Giuseppe Zanotti, Paul M. Horowitz

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

The NH2-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., Drainer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P.M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-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 Δ1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-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 NH2 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.

Original languageEnglish (US)
Pages (from-to)13938-13947
Number of pages10
JournalJournal of Biological Chemistry
Volume274
Issue number20
DOIs
StatePublished - May 14 1999
Externally publishedYes

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Thiosulfate Sulfurtransferase
Crystal structure
Crystals
Molecular Chaperones
X ray crystallography
X Ray Crystallography
Crystallization
Rigidity
Contacts (fluid mechanics)
Escherichia coli
Conformations
Molten materials
Catalytic Domain
Stabilization
Adenosine Triphosphate
Fluorescence
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions. / Trevino, Richard J.; Gliubich, Francesca; Berni, Rodolfo; Cianci, Michele; Chirgwin, John; Zanotti, Giuseppe; Horowitz, Paul M.

In: Journal of Biological Chemistry, Vol. 274, No. 20, 14.05.1999, p. 13938-13947.

Research output: Contribution to journalArticle

Trevino, Richard J. ; Gliubich, Francesca ; Berni, Rodolfo ; Cianci, Michele ; Chirgwin, John ; Zanotti, Giuseppe ; Horowitz, Paul M. / NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 20. pp. 13938-13947.
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abstract = "The NH2-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., Drainer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P.M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-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 Δ1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-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 NH2 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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AU - Trevino, Richard J.

AU - Gliubich, Francesca

AU - Berni, Rodolfo

AU - Cianci, Michele

AU - Chirgwin, John

AU - Zanotti, Giuseppe

AU - Horowitz, Paul M.

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N2 - The NH2-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., Drainer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P.M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-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 Δ1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-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 NH2 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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