Reversible folding of rhodanese. Presence of intermediate(s) at equilibrium.
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.
Tandon
S
S
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Horowitz
P M
PM
D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D009391:Nephelometry and Turbidimetry; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013466:Sulfurtransferases / Q000378:metabolism*; D013816:Thermodynamics; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*
D006146:Guanidines; D046911:Macromolecular Substances; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D019791:Guanidine; D004229:Dithiothreitol