title: Characterization of a molten globule intermediate during GdnHCl-induced unfolding of RTEM beta-lactamase from Escherichia coli.
abstract: GdnHCl-induced unfolding and reversible folding of beta-lactamase from E. coli have been investigated by measuring enzymatic activity, fluorescence emission and far-UV circular dichroism as indices of the extent of denaturation. The non-coincidence of far-UV CD and fluorescence data and existence of an inflection point clearly suggest the presence of an equilibrium intermediate. The existence of the equilibrium intermediate at around 1 M is corroborated by its enhanced binding of fluorophobic probe 1,8-ANS. The intermediate was found to have a compact shape as measured by its Stokes radius by size-exclusion chromatography. Furthermore, near-UV CD analysis of this enzymatically inactive intermediate showed a significantly disrupted tertiary structure with only a minor change in the secondary structure, which is a characteristic of typical molten globule states. Estimation of the activation energy from the kinetics of unfolding of the protein monitored by fluorescence and CD suggests that the intermediate may be separated from the native and the unfolded state by a high activation-energy barrier.
authors.affiliation: Department of Biophysics, Molecular Biology and Genetics, University College of Science, Calcutta, India.
authors.forename: D
authors.initials: D
authors.lastname: Sarkar
authors.forename: C
authors.initials: C
authors.lastname: DasGupta
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006146:Guanidines / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D009391:Nephelometry and Turbidimetry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D001618:beta-Lactamases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
chemical_list: D000817:Anilino Naphthalenesulfonates; D006146:Guanidines; D001618:beta-Lactamases