TEM-1 beta-lactamase folds in a nonhierarchical manner with transient non-native interactions involving the C-terminal region.
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.
Lejeune
Annabelle
A
Laboratoire d'Enzymologie and Laboratoire de Cristallographie des Protéines, Centre for Protein Engineering, Université de Liège, Institut de Chimie B6, 4000 Liège (Sart Tilman), Belgium.
Pain
Roger H
RH
Charlier
Paulette
P
Frère
Jean-Marie
JM
Matagne
André
A
D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001618:beta-Lactamases; C034413:beta-lactamase TEM-1