Biophysical characterization of the interaction of the β-lactamase TEM-1 with its protein inhibitor BLIP

BLIP is a secreted protein from Streptomyces clavuligerus that inhibits a wide range of beta-lactamases. Here we investigate the tight interaction of BLIP, expressed heterologousely in E. coli, with TEM-1. Kinetic and thermodynamic constants were determined using methods with the proteins either in a homogeneous or in a heterogeneous phase. While values of Delta Delta G((mut-wt)) are similar whether measured by fluorescence quench, enzyme inhibition, or surface plasmon resonance, absolute values of Delta G and kinetic constants vary. Association and dissociation rate constants of 10(5) M-1 s(-1) and 10(-4) s(-1) respectively, and a nanomolar affinity were determined for the wild-type proteins. The highest affinity is measured at pH 7.5, with a decreasing association rate constant at higher pH values, and an increasing dissociation rate constant at lower pH values. The marginal effect of salt on the kinetics of binding, as well as the calculated surface potentials, suggests a limited role for electrostatic forces in guiding this reaction. Still, mutations of interfacial residues affect the rate of association significantly, so that an increase in the net negative charge on either protein reduces the association rate constant. We show that simple electrostatic rules can explain this behavior. BLIP inhibits the catalytic activity of TEM-1 by binding its active site. Yet, mutations of active site residues on TEM-1 only have a moderate though cooperative effect on the binding energy. This can be explained in light of the peripheral location of the active site in the interface between the two proteins.