Investigation of the folding pathway of the TEM‐1 β‐lactamase

Abstract

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.

Folding intermediates of β‐lactamase recognized by GroEL

“…The equilibrium folding of ß-lase, both from Staphylococcus aureus [12] and from E. coli [11], follows a three-state behavior. The refolding kinetics reveal the existence of three phases and thus at least two transient intermediates (Fig.…”

“…Identical experiments were performed in the absence of GroEL. The folding of the enzyme is biphasic [11] and can be given [18] by…”

“…2, using the amplitudes from Figs. 1A and 1C and taking into account that the denaturant already inhibits the reaction at a low concentration (0.1 M) and can be considered a competitive inhibitor (ATi = 0.38 M) [11], the fast folding rate constants for the regain of enzymatic activity were obtained. For the wild-type, a rate constant of k-5.0 X 10~2 s _1 and for the Cys-Ala mutant a rate constant of k = 4.7x 10~2 s _1 was de- termined.…”

“…The precursor of ß-lase is known to bind very tightly [10], while the mature form binds much more weakly [7], and differences between reduced and oxidized ß-lase are also apparent (this study). The thermodynamics and kinetics of ß-lase folding have been extensively characterized [7,[10][11][12][13]. ß-Lase is able to fold to the native state both in the cytoplasm and in the periplasm, and it does not need its disulfide bond to reach the native state [14], which can thus serve as an optional constraint in the structure.…”

Protein–protein binding affinities by pulse proteolysis: Application to TEM‐1/BLIP protein complexes

“…The folding pathway of TEM-1 has been described in some detail and is multistate. 8,9 In this respect, it presents a nontrivial and realistic test of the method, as many interesting protein interactions are not composed of small, wellbehaved, two-state-folding proteins. We propose pulse proteolysis as a high-throughput method for evaluating complex stabilities for a wide variety of protein-protein interactions.…”