Folding of an Abridged β-Lactamase

Abstract

The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B. licheniformis exo small β−lactamase (ES-βL) have been measured. ES-βL lacking 19 residues (ES-βLCΔ19) has no enzymic activity. Deletion of the last 14 residues produces ES-βLCΔ14, which is 0.1% active. The enzyme lacking nine residues (ES-βLCΔ9) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme. Although ES-βLCΔ9 folds properly, it does so 4 orders of magnitude slower than ES-βL, making possible the isolation and characterization of a compact intermediate state (IP ES-βLCΔ9). Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between IP ES-βLCΔ9 and other intermediate slow folding. Residues removed in ES-βLCΔ9 and ES-βLCΔ14 are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence. The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level. The results are compared with other examples of this kind in the folding literature.