title: 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.
authors.affiliation: Biophysics Graduate Group, University of California, Berkeley, Berkeley, California 94729, USA.
authors.forename: Melinda S
authors.initials: MS
authors.lastname: Hanes
authors.forename: Kathleen
authors.initials: K
authors.lastname: Ratcliff
authors.forename: Susan
authors.initials: S
authors.lastname: Marqusee
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Handel
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D014508:Urea / Q000737:chemistry / Q000494:pharmacology; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D050505:Mutant Proteins; C065596:beta-lactamase-inhibitor protein, Streptomyces; D014508:Urea; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1