Protein-protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes

Abstract

Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein-ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein-protein complex involving the β-lactamase TEM-1 and various β-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C(m), of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein-protein complexes. From a small set (n = 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC(m) was observed. From this "calibration curve," accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived ΔC(m) values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies.

Figure 1

Pulse proteolysis denaturation experiments analyzed on 15% SDS polyacrylamide gels for (A) TEM-1 and (B) TEM-1 with BLIP. Urea concentrations are listed above each lane, and molecular weights for ladder proteins are listed to the left of each gel, with an arrow pointing at the proteins of interest (TEM-1, 29 kDa or BLIP, 17 kDa). The remaining bands are due to thermolysin (35 kDa) and its autolysis products (23 and 12 kDa). (C) Denaturation curves for TEM-1 (solid squares), TEM-1 with BLIP (open squares), or BLIP (open triangles) fitted to a Boltzmann sigmoid to determine the inflection point, 2.00M and 4.49M urea for TEM-1 and BLIP, respectively. (D) Varying the pulse length from 1–10 min at a urea concentration equal to the C_m has little effect on native TEM-1.