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


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., Dramer, 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 non-native 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 Delta1-7 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. Study holds ProTherm entries: 13151, 13152, 13153, 13154, 13155, 13156, 13157, 13158 Extra Details: beta-mercaptoethanol(200 mM) was added in the experiment stability; truncated rhodanese; molten globule state

Submission Details

ID: NqHG34SV4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
Trevino RJ;Gliubich F;Berni R;Cianci M;Chirgwin JM;Zanotti G;Horowitz PM,J. Biol. Chem. (1999) NH2-terminal sequence truncation decreases the stability of bovine rhodanese, minimally perturbs its crystal structure, and enhances interaction with GroEL under native conditions. PMID:10318804
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1RHS 1998-01-21 1.36 SULFUR-SUBSTITUTED RHODANESE
2ORA 1996-08-01 1.99 RHODANESE (THIOSULFATE: CYANIDE SULFURTRANSFERASE)
1ORB 1995-10-15 2.0 ACTIVE SITE STRUCTURAL FEATURES FOR CHEMICALLY MODIFIED FORMS OF RHODANESE
1DP2 2000-12-13 2.01 CRYSTAL STRUCTURE OF THE COMPLEX BETWEEN RHODANESE AND LIPOATE
1BOI 1999-04-27 2.2 N-TERMINALLY TRUNCATED RHODANESE
1BOH 1999-04-27 2.3 SULFUR-SUBSTITUTED RHODANESE (ORTHORHOMBIC FORM)
1RHD 1978-01-16 2.5 STRUCTURE OF BOVINE LIVER RHODANESE. I. STRUCTURE DETERMINATION AT 2.5 ANGSTROMS RESOLUTION AND A COMPARISON OF THE CONFORMATION AND SEQUENCE OF ITS TWO DOMAINS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.9 Thiosulfate sulfurtransferase P46635 THTR_CRIGR
91.9 Thiosulfate sulfurtransferase P24329 THTR_RAT
100.0 Thiosulfate sulfurtransferase P00586 THTR_BOVIN