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Membrane Proteins

Volume 1063 of the series Methods in Molecular Biology pp 37-56

Date:

Measuring Transmembrane Helix Interaction Strengths in Lipid Bilayers Using Steric Trapping

  • Heedeok HongAffiliated withDepartment of Chemistry, Michigan State UniversityDepartment of Biochemistry & Molecular Biology, Michigan State University
  • , Yu-Chu ChangAffiliated withDepartment of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, University of California
  • , James U. BowieAffiliated withDepartment of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, University of California

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Abstract

We have developed a method to measure strong transmembrane (TM) helix interaction affinities in lipid bilayers that are difficult to measure by traditional dilution methods. The method, called steric trapping, couples dissociation of biotinylated TM helices to a competitive binding by monovalent streptavidin (mSA), so that dissociation is driven by the affinity of mSA for biotin and mSA concentration. By adjusting the binding affinity of mSA through mutation, the method can obtain dissociation constants of TM helix dimers (K d,dimer) over a range of six orders of magnitudes. The K d,dimer limit of measurable target interaction is extended 3–4 orders of magnitude lower than possible by dilution methods. Thus, steric trapping opens up new opportunities to study the folding and assembly of α-helical membrane proteins in lipid bilayer environments. Here we provide detailed methods for applying steric trapping to a TM helix dimer.

Key words

Membrane protein folding Steric trap Glycophorin A dimer Transmembrane helix interaction Monovalent streptavidin Biotinylation