Comparison of Protein‐Protein Interactions between BLIP and Class A Beta‐Lactamases

Abstract

The goal of our project is to determine and compare the binding thermodynamics of the interaction between β‐lactamase Inhibitor Protein (BLIP) mutants and Class A β‐Lactamases using ITC. We have previously determined the thermodynamics of binding interactions between TEM‐1, class A β‐lactamase family, and a set of alanine substituted contact residue mutants of BLIP (Wang J., et al. JBC. 2007; 282:17676–84). We have extended our approach to other members of class A β‐lactamases. Here, we report the binding thermodynamics between the inactive TEM‐1 mutant (S70A) and BLIP. We find that the alanine substitution of the active site Ser70 of TEM‐1 results in the lowering of the iso‐enthalpic temperature, which is more enthalpically favorable. The binding between BLIP and Bla1, another class A β‐lactamase, is dominated by an entropy driven force with an extrapolated iso‐enthalpic temperature of 42oC. Its binding heat capacity change is ∼ −450 cal/mol.K (∼ 20% less negative than that of BLIP/TEM‐1). However, the BLIP mutant W150A interaction with Bla1 shows a non‐linear relationship between the binding enthalpy and temperature with a positive binding heat capacity change at low temperature and a negative binding heat capacity change at high temperature, which is contrary to the general observation that the heat capacity change is negative for protein‐protein interactions.Support from AHA & UH to D.C. and NIH AI32956 to T.P.