Alteration around the active site of rhodanese during urea-induced denaturation and its implications for folding.
The enzyme rhodanese contains two globular domains connected by a tether region and associated by strong hydrophobic interactions. The protein has proven to be very difficult to refold without assistance to prevent oxidation and aggregation. For this study, the active site cysteine 247, near the interdomain region, was modified with the environmentally sensitive fluorescent probe, 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS), to yield a derivative that reversibly unfolds. Structural transitions during urea unfolding/refolding were complex and multiphasic. Increasing urea concentrations increased the IAANS fluorescence intensity and polarization. Both values reached maxima at approximately 4 m urea, where there is a concomitant large exposure of hydrophobic sites as reported by both IAANS and the noncovalent fluorescent probe, bis-ANS. The exposure of the hydrophobic sites arises from the decrease in strong interaction between the domain interfaces, which lead to their partial separation. This correlates with the loss of activity of the unlabeled enzyme. Above 4.5 m urea, there is progressive loss of rigid, hydrophobic surfaces, and both fluorescence and polarization of IAANS decrease, with accompanying loss of secondary structure. These results are consistent with a folding model in which there is an initial, rapid hydrophobic collapse of the denatured form to an intermediate with native like secondary structure, with exposed interdomain, hydrophobic surfaces. This step is followed by adjustment of the domain-domain interactions and the proper positioning of reduced cysteine 247 at the active site.
Bhattacharyya
A M
AM
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284, USA.
Horowitz
P
P
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D003545:Cysteine; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009282:Naphthalenesulfonates; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; C011488:IAANS; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014508:Urea; D013884:Thiosulfate Sulfurtransferase; D003545:Cysteine