Acid-induced partly folded conformation resembling a molten globule state of xylanase from an alkalothermophilic Bacillus sp.
Nonnative protein structures having a compact secondary, but not rigid tertiary structure, have been increasingly observed as intermediate states in protein folding. We have shown for the first time during acid-induced unfolding of xylanase (Xyl II) the presence of a partially structured intermediate form resembling a molten globule state. The conformation and stability of Xyl II at acidic pH was investigated by equilibrium unfolding methods. Using intrinsic fluorescence and CD spectroscopic studies, we have established that Xyl II at pH 1.8 (A-state) retains the helical secondary structure of the native protein at pH 7.0, while the tertiary interactions are much weaker. At variance, from the native species (N-state), Xyl II in the A-state binds 1-anilino-8-sulfonic acid (ANS) indicating a considerable exposure of aromatic side chains. Lower concentration of Gdn HCl are required to unfold the A-state. For denaturation by Gdn HCl, the midpoint of the cooperative unfolding transition measured by fluorescence for the N-state is 3.5 +/- 0.1 M, which is higher than the value (2.2 +/- 0.1 M) observed for the A-state at pH 1.8. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its structural stability that is characteristic for native proteins.
Nath
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D
Biochemical Sciences Division, National Chemical Laboratory, Pashan Pune 411008, India.
Rao
M
M
D020106:Acrylamide / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D019791:Guanidine