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 4 8 degrees C, was partially digested with trypsin, endoproteinase Glu-C , or thermolysin. Peptides were analyzed by matrix-assisted laser deso rption and ionization mass spectrometry and aligned with the known seq uence. From the protease cleavage sites which become protected upon bi nding 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 u ncovered by the slight dislocation of other structural elements. This explains how hydrophobic surface on the substrate protein can become a ccessible while scarcely disrupting the hydrogen bond network of the n ative structure. An analysis of the GroEL-bound peptides bound after d igestion of the beta-lactamase showed no obvious sequence motifs, indi cating that binding is provided by hydrophobic patches in the three-di mensional structure.