Identification of the binding surface on beta-lactamase for GroEL by limited proteolysis and MALDI-mass spectrometry.

Escherichia coli beta-lactamase, alone or as a complex with GroEL at 48 degreesC, was partially digested with trypsin, endoproteinase Glu-C, or thermolysin. Peptides were analyzed by matrix-assisted laser desorption and ionization mass spectrometry and aligned with the known sequence. From the protease cleavage sites which become protected upon binding and those which become newly accessible, a model of the complex is proposed in which the carboxy-terminal helix has melted, two loops form the binding interface and the large beta-sheet become partially uncovered by the slight dislocation of other structural elements. This explains how hydrophobic surface on the substrate protein can become accessible while scarcely disrupting the hydrogen bond network of the native structure. An analysis of the GroEL-bound peptides bound after digestion of the beta-lactamase showed no obvious sequence motifs, indicating that binding is provided by hydrophobic patches in the three-dimensional structure.

Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins.

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.

Two conformational states of beta-lactamase bound to GroEL: a biophysical characterization.

Escherichia coli RTEM beta-lactamase, in which both cysteine residues which form the single disulfide bond have been mutated to alanine residues, can form stable reversible complexes with GroEL under two different sets of conditions. Starting with the GdmCl-denatured enzyme, it is bound to GroEL in a state where no protons are protected against exchange with 2H2O, as determined by electrospray ionization mass spectrometry (ESI-MS). In contrast, when native protein is destabilized at high temperature and added to GroEL, a conformation is bound with 18 protected protons after two hours of exchange. While the high-temperature complex can form both with the wild-type enzyme (with intact disulfide bond) and the Cys-Ala double mutant, only the latter protein can form a complex starting from GdmCl denatured states. Thus, two different sets of conformations of the same protein can be bound, depending both on the conditions used to form the complex and on the intrinsic stability of the intermediate recognized by GroEL, and we have characterized the properties of both complexes.

Folding intermediates of beta-lactamase recognized by GroEL.

beta-Lactamase, from which the disulfide bond was removed by two Cys-->Ala mutations, forms stable complexes with GroEL only during the first 30 s of folding, while wild-type beta-lactamase forms no stable complex under these conditions. The 3-phasic kinetics of folding are very similar between wild-type and mutant. After 4 s, Trp-210 is already juxtaposed to the disulfide bond, but proline cis-trans isomerization has not yet taken place and almost no enzymatic activity is observed. This shows that GroEL is unable to bind late folding intermediates and also discriminates between the degree of unfolding possible in wild-type disulfide-containing beta-lactamase and the Cys-Ala mutant.

beta-Lactamase binds to GroEL in a conformation highly protected against hydrogen/deuterium exchange.

Escherichia coli RTEM beta-lactamase reversibly forms a stable complex with GroEL, devoid of any enzymatic activity, at 48 degrees C. When beta-lactamase is diluted from this complex into denaturant solution, its unfolding rate is identical to that from the native state, while the unfolding rate from the molten globule state is too fast to be measured. Electrospray mass spectrometry shows that the rate of proton exchange in beta-lactamase in the complex at 48 degrees C is slower than in the absence of GroEL at the same temperature, and resembles the exchange of the native state at 25 degrees C. Similarly, the final number of protected deuterons is higher in the presence of GroEL than in its absence. We conclude that, for beta-lactamase, a state with significant native structure is bound to GroEL. Thus, different proteins are recognized by GroEL in very different states, ranging from totally unfolded to native-like, and this recognition may depend on which state can provide sufficient accessible hydrophobic amino acids in a suitably clustered arrangement. Reversible binding of native-like states with hydrophobic patches may be an important property of GroEL to protect the cell from aggregating protein after heat-shock.