Measuring the Thermodynamic Stability of Strong Protein-Protein Interactions in Lipid Bilayers Using a Steric Trap

Abstract

Protein-protein interactions within cell membranes play crucial roles in the assembly of membrane proteins and cell signaling. Although thermodynamic analysis of binding affinity is essential for understanding the stability, specificity, and function of membrane proteins, these measurements can be difficult to make for high affinity interactions in lipid bilayers. To address this problem, we have developed a steric trap method, which couples the dissociation of a membrane protein complex to another measurable binding event. The method postulates that a concomitant binding of two bulky monovalent streptavidins (mSA) to a doubly biotinylated protein complex occur only when the protein is dissociated due to the steric hindrance. This leads to an attenuated binding affinity of the second mSA, which is directly correlated to the stability of a target interaction. We tested the method using a glycophorinA transmembrane domain fusion to staphylococcal nuclease (SNGpA), which forms a stable dimer in various lipid environments. Equilibrium binding of mSA to the enzymatically biotinylated SNGpA exhibited two distinctive phases, which corresponds to the tight first mSA binding and the weaker second binding in decyl maltoside (DM) micelles and palmitoyloleoyl phophatidylcholine (POPC) bilayers. The stability of GpA dimer extracted from the second binding event at different micellar concentrations yielded the dissociation constants (Kd) of 10−8∼10−7 M, which agree well with the previous results. The stability of GpA dimer is enhanced in POPC bilayers by ∼4 orders of magnitude at comparable mole fractions. The difference free energies between wild-type and destabilized mutants in both systems correlate with the equilibrium sedimentation data measured in C8E5 micelles. Our results suggest that the steric trap method provides a powerful tool to study the strong protein-protein interactions in lipid bilayers.