Biomaterial Database

Characterization of a molten globule intermediate during GdnHCl-induced unfolding of RTEM beta-lactamase from Escherichia coli. 1996

D Sarkar, and C DasGupta

Department of Biophysics, Molecular Biology and Genetics, University College of Science, Calcutta, India.

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D009391	Nephelometry and Turbidimetry	Chemical analysis based on the phenomenon whereby light, passing through a medium with dispersed particles of a different refractive index from that of the medium, is attenuated in intensity by scattering. In turbidimetry, the intensity of light transmitted through the medium, the unscattered light, is measured. In nephelometry, the intensity of the scattered light is measured, usually, but not necessarily, at right angles to the incident light beam.	Turbidimetry,Nephelometry,Turbidimetry and Nephelometry
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D011489	Protein Denaturation	Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein.	Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D002942	Circular Dichroism	A change from planar to elliptic polarization when an initially plane-polarized light wave traverses an optically active medium. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Circular Dichroism, Vibrational,Dichroism, Circular,Vibrational Circular Dichroism
D004789	Enzyme Activation	Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.	Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D004795	Enzyme Stability	The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat.	Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006146	Guanidines	A family of iminourea derivatives. The parent compound has been isolated from mushrooms, corn germ, rice hulls, mussels, earthworms, and turnip juice. Derivatives may have antiviral and antifungal properties.
D000817	Anilino Naphthalenesulfonates	A class of organic compounds which contain an anilino (phenylamino) group linked to a salt or ester of naphthalenesulfonic acid. They are frequently used as fluorescent dyes and sulfhydryl reagents.	Naphthalenesulfonates, Anilino