Folding of an abridged beta-lactamase.
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.
Santos
Javier
J
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 180, (1876) Bernal, Buenos Aires, Argentina.
Gebhard
Leopoldo G
LG
Risso
Valeria A
VA
Ferreyra
Raul G
RG
Rossi
Juan P F C
JP
Ermácora
Mario R
MR
D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics*
D001426:Bacterial Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D001618:beta-Lactamases; D019791:Guanidine; D003545:Cysteine