enzyme kinetics; beta-lactamase; molecular modelling; protein stability; therml unfolding
Abstract :
[en] The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and spectroscopic properties of the mutant enzyme did not differ significantly from those of the wild type, indicating that the mutation has only a very limited effect on the structure of the protein. The stability of the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globule intermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290 becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminal region of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seen in the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observed with the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290. This residue is suggested to be buried in a nonpolar environment from which it has to escape during subsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in which the C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with a nonhierarchical folding process.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Lejeune, Annabelle ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Pain, R. H.
Charlier, Paulette ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie
Language :
English
Title :
Tem-1 Beta-Lactamase Folds in a Nonhierarchical Manner with Transient Non-Native Interactions Involving the C-Terminal Region
Publication date :
29 January 2008
Journal title :
Biochemistry
ISSN :
0006-2960
eISSN :
1520-4995
Publisher :
American Chemical Society, Washington, United States - District of Columbia
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