caa a22 4500 475744500 CHVBK 20180406123509.0 cr unu---uuuuu 170329e20000701xx s 000 0 eng 10.1023/A:1026491615076 doi (NATIONALLICENCE)springer-10.1023/A:1026491615076 Active-Site Sulfhydryl Chemistry Plays a Major Role in the Misfolding of Urea-Denatured Rhodanese [Elektronische Daten] [Markandeswar Panda, Paul Horowitz] Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activity upon refolding due to the formation of aggregates and disulfide-linked misfolded states unless a large excess of reductant such as 200 mM β-ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Chem. 265, 5967]. Even then, refolding is incomplete. We have studied the unfolding and refolding of three rhodanese forms whose crystal structures are known: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the active site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in solution differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimum three-state folding model of CMR (N↔I↔U) followed by fluorescence at 363 nm, (N↔I) by fluorescence at 318 nm, and CD (I↔U) is consistent with the presence of a thermodynamically stable molten globule intermediate in 5-6 M urea. We conclude that the active-site sulfhydryl group in the persulfide form is very reactive; therefore, its modification leads to the successful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete reversibility of CMR suggests that oxidation among the three non-active-site SH groups can represent a minor trap for refolding through species that can be easily reduced. Plenum Publishing Corporation, 2000 Rhodanese nationallicence protein folding nationallicence active site nationallicence urea denaturation nationallicence molten globule nationallicence Panda Markandeswar Department of Biochemistry, University of Texas Health Science Center at San Antonio, 78229-3900, San Antonio, Texas aut Horowitz Paul Department of Biochemistry, University of Texas Health Science Center at San Antonio, 78229-3900, San Antonio, Texas aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/5(2000-07-01), 399-409 0277-8033 19:5<399 2000 19 10930 https://doi.org/10.1023/A:1026491615076 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1026491615076 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Panda Markandeswar Department of Biochemistry, University of Texas Health Science Center at San Antonio, 78229-3900, San Antonio, Texas aut NATIONALLICENCE 700 1- Horowitz Paul Department of Biochemistry, University of Texas Health Science Center at San Antonio, 78229-3900, San Antonio, Texas aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/5(2000-07-01), 399-409 0277-8033 19:5<399 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer