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Investigation of the folding pathway of the TEM‐1 β‐lactamase
Author(s) -
Vanhove Marc,
Raquet Xavier,
Frère JeanMarie
Publication year - 1995
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340220204
Subject(s) - molten globule , circular dichroism , native state , guanidine , chemistry , denaturation (fissile materials) , protein secondary structure , globular protein , protein folding , crystallography , equilibrium unfolding , protein tertiary structure , intermediate state , folding (dsp implementation) , isomerization , hydrochloride , stereochemistry , organic chemistry , biochemistry , catalysis , philosophy , theology , electrical engineering , nuclear chemistry , engineering
The TEM‐1 β‐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. Thisstate 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 denaturedprotein. 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 experimentsrevealed the existence of a transient intermediate conformation between thethermodynamically 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 β‐lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probablyreflect a very complex process kinetically limited by proline cis/transisomerization. © 1995 Wiley‐Liss, Inc.