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Kinetic and thermodynamic consequences of the removal of the Cys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase

Vanhove, Marc and Guillaume, Gilliane and Ledent, Philippe and Richards, John H. and Pain, Roger H. and Frére, Jean-Marie (1997) Kinetic and thermodynamic consequences of the removal of the Cys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase. Biochemical Journal, 321 (Part 2). pp. 413-417. ISSN 0264-6021. https://resolver.caltech.edu/CaltechAUTHORS:20150427-161237094

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Abstract

Class A β-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 β-lactamase, this bond was removed by introducing a Cys-77 Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N H U). Both the folded mutant protein (N) and, to a lesser extent, the thermodynamically stable intermediate, H, were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.


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http://www.biochemj.org/bj/321/bj3210413.htmPublisherArticle
Additional Information:© 1997 The Biochemical Society, London. Received 17 April 1996/4 September 1996; Accepted 18 September 1996. This work was supported, in part, by the Belgian Government in the frame of a Po#le d'Attraction Interuniversitaire (PAI no. 19), an Action Concerte!e with the Belgian Government (convention 89/94-130) and the Fonds de la Recherche Scienti®que Medicale (contract no. 3.4537.88). M. V. is a fellow of the Fond pour la Formation a' la Recherche dans l 'Industrie et l'Agriculture (F.R.I.A., Brussels, Belgium).
Funders:
Funding AgencyGrant Number
Pôle d'Attraction InteruniversitairePAI no. 19
Action Concertéeconvention 89/94-130
Fonds de la Recherche Scientifique Medicale3.4537.88
Fond pour la Formation à la Recherche dans l 'Industrie et l'Agriculture (FRIA)UNSPECIFIED
Issue or Number:Part 2
Record Number:CaltechAUTHORS:20150427-161237094
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150427-161237094
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:57027
Collection:CaltechAUTHORS
Deposited By: Joanne McCole
Deposited On:28 Apr 2015 18:42
Last Modified:03 Oct 2019 08:20

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