Method to measure strong protein-protein interactions in lipid bilayers using a steric trap.
Measuring high affinity protein-protein interactions in membranes is extremely challenging because there are limitations to how far the interacting components can be diluted in bilayers. Here we show that a steric trap can be employed for stable membrane interactions. We couple dissociation to a competitive binding event so that dissociation can be driven by increasing the affinity or concentration of the competitor. The steric trap design used here links monovalent streptavidin binding to dissociation of biotinylated partners. Application of the steric trap method to the well-characterized glycophorin A transmembrane helix (GpATM) reveals a dimer that is dramatically stabilized by 4-5 kcal/mol in palmitoyloleoylphosphatidylcholine bilayers compared to detergent. We also find larger effects of mutations at the dimer interface in bilayers compared to detergent suggesting that the dimer is more organized in a membrane environment. The high affinity we measure for GpATM in bilayers indicates that a membrane vesicle many orders of magnitude larger than a bacterial cell would be required to measure the dissociation constant using traditional dilution methods. Thus, steric trapping can open new biological systems to experimental scrutiny in natural bilayer environments.
Department of Chemistry and Biochemistry, University of California, Los Angeles-Department of Energy Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095, USA.
Heedeok
H
Hong
Tracy M
TM
Blois
Zheng
Z
Cao
James U
JU
Bowie
D003902:Detergents / Q000737:chemistry; D008051:Lipid Bilayers / Q000378:metabolism*; D008823:Micelles; D008958:Models, Molecular; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D055550:Protein Stability
D003902:Detergents; D008051:Lipid Bilayers; D008823:Micelles