title: Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures.
abstract: Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.
authors.affiliation: Department of Chemistry, University of California, Santa Cruz 95064.
authors.forename: W D
authors.initials: WD
authors.lastname: Anderson
authors.forename: A L
authors.initials: AL
authors.lastname: Fink
authors.forename: L J
authors.initials: LJ
authors.lastname: Perry
authors.forename: R
authors.initials: R
authors.lastname: Wetzel
mesh_terms: D003432:Cross-Linking Reagents; D004220:Disulfides; D007700:Kinetics; D009113:Muramidase / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D013050:Spectrometry, Fluorescence; D013604:T-Phages / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D014364:Tryptophan
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D014364:Tryptophan; D009113:Muramidase