Reversible folding of rhodanese. Presence of intermediate(s) at equilibrium.


Abstract

For the first time completely reversible unfolding was achieved for guanidinium chloride-denatured rhodanese using a systematically defined protocol. These conditions included beta-mercaptoethanol, lauryl maltoside, and sodium thiosulfate. All components were required to get more than the previous best reactivation with lauryl maltoside of 17% (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15681). Non-coincidental transition curves were obtained by monitoring different parameters including: (i) variation in the activity, (ii) shifts of the fluorescence wavelength maximum, and (iii) variation in ellipticity at 220 nm. The transition followed by the fluorescence wavelength maximum was asymmetric and resolvable into two separate transitions. A thermodynamic analysis was used to define the energetics of the two processes. Studies with the fluorescent "apolar" probe 1,8ANS are consistent with the appearance of organized hydrophobic surfaces following the first transition. Near UV CD measurements indicated that the first transition is associated with a loss of dyssymmetry around at least some of the tryptophans. Thus, the unfolding of rhodanese is complex, and there are detectable intermediate(s) during the process. These results suggest that reversible unfolding occurs in two discrete stages: 1) loss of tertiary interactions and activity, with retention of secondary structure, and 2) loss of secondary structure. The available x-ray structure suggests that the first transition can be associated with changes in the domain interactions, which may modulate the effectiveness of helix dipoles in lowering the pKa of the active site sulfhydryl. Study holds ProTherm entries: 5166 Extra Details: sodium thiosulfate(50 mM) was added in the experiment

Submission Details

ID: iWvc9Xzg

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Tandon S;Horowitz PM,J. Biol. Chem. (1989) Reversible folding of rhodanese. Presence of intermediate(s) at equilibrium. PMID:2722881
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