Folding of an abridged beta-lactamase

Biochemistry. 2004 Feb 17;43(6):1715-23. doi: 10.1021/bi0358162.

Abstract

The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B. licheniformis exo small beta-lactamase (ES-betaL) have been measured. ES-betaL lacking 19 residues (ES-betaL(C)(Delta)(19)) has no enzymic activity. Deletion of the last 14 residues produces ES-betaL(C)(Delta)(14), which is 0.1% active. The enzyme lacking nine residues (ES-betaL(C)(Delta)(9)) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme. Although ES-betaL(C)(Delta)(9) folds properly, it does so 4 orders of magnitude slower than ES-betaL, making possible the isolation and characterization of a compact intermediate state (I(P) ES-betaL(C)(Delta)(9)). Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between I(P) ES-betaL(C)(Delta)(9) and other intermediate slow folding. Residues removed in ES-betaL(C)(Delta)(9) and ES-betaL(C)(Delta)(14) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus / enzymology
  • Bacillus / genetics
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics*
  • Circular Dichroism
  • Cysteine / genetics
  • Guanidine / chemistry
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Kinetics
  • Mutagenesis, Site-Directed
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Protein Denaturation / genetics
  • Protein Folding*
  • Protein Structure, Secondary / genetics
  • Sequence Deletion*
  • Spectrometry, Fluorescence
  • beta-Lactamases / chemistry*
  • beta-Lactamases / genetics*

Substances

  • Bacterial Proteins
  • Isoenzymes
  • Peptide Fragments
  • beta-Lactamases
  • Guanidine
  • Cysteine