title: Role of a disulfide cross-link in the conformational stability of a thermostable xylanase.
abstract: The role of a S-S cross-link in the conformational stability of xylanase from Humicola lanuginosa has been investigated using CD, UV absorption spectroscopy, and RIA displacement studies. Our studies show that reduction and carboxymethylation of the S-S cross-link in xylanase results in a gross conformational perturbation of the protein. The secondary structure analysis of the CD spectra indicates that the xylanase with an intact S-S contains 66% beta-sheet structure and remaining random coil. Cleavage of the S-S bond results in a loss of 25% beta-sheet structure. Thermal denaturation studies using CD spectroscopy and pH-dependent tyrosine ionization studies using UV spectroscopy show that the presence of disulfide cross-link offers resistance against unfolding by extremes of temperature and pH. Further, we demonstrate that the heat-induced changes in xylanase with intact S-S bond are almost totally reversible, while those in the S-S cleaved enzyme fail to show any significant reversal. Our studies support the present theory that S-S cross-links exert their stabilizing effect in proteins by destabilizing the unfolded state of the protein and forcing it back to a more folded state.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore.
authors.forename: U
authors.initials: U
authors.lastname: Tatu
authors.forename: S K
authors.initials: SK
authors.lastname: Murthy
authors.forename: P J
authors.initials: PJ
authors.lastname: Vithayathil
mesh_terms: D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis*; D004795:Enzyme Stability / Q000502:physiology; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006358:Hot Temperature; D008745:Methylation; D003904:Mitosporic Fungi / Q000201:enzymology; D011487:Protein Conformation; D011863:Radioimmunoassay; D013056:Spectrophotometry, Ultraviolet; D043325:Xylan Endo-1,3-beta-Xylosidase
chemical_list: D004220:Disulfides; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase