Investigation of the folding pathway of the TEM-1 beta-lactamase.
The TEM-1 beta-lactamase is a globular protein containing 12 proline residues. The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism. The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state. This state is 5.2 +/- 0.4 kcal/mol less stable than the native conformation and 5.7 +/- 0.2 kcal/mol more stable than the fully denatured protein. This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the "molten globule" is discussed. Refolding kinetic experiments revealed the existence of a transient intermediate conformation between the thermodynamically stable intermediate and the native protein. This transient intermediate appears rapidly during the folding reaction. It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation. The final refolding step of the TEM-1 beta-lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probably reflect a very complex process kinetically limited by proline cis/trans isomerization.
Vanhove
M
M
Laboratoire d'Enzymologie, Institut de Chimie, B6 Université de Liège, Sart-Tilman, Belgium.
Raquet
X
X
Frère
J M
JM
D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
D006146:Guanidines; D014364:Tryptophan; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D019791:Guanidine