Membrane Proteins pp 37-56

Part of the Methods in Molecular Biology book series (MIMB, volume 1063)

Measuring Transmembrane Helix Interaction Strengths in Lipid Bilayers Using Steric Trapping

  • Heedeok Hong
  • Yu-Chu Chang
  • James U. Bowie
Protocol

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 (Kd,dimer) over a range of six orders of magnitudes. The Kd,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 

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Heedeok Hong
    • 1
    • 2
  • Yu-Chu Chang
    • 3
  • James U. Bowie
    • 3
  1. 1.Department of ChemistryMichigan State UniversityEast LansingUSA
  2. 2.Department of Biochemistry & Molecular BiologyMichigan State UniversityEast LansingUSA
  3. 3.Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology InstituteUniversity of CaliforniaLos AngelesUSA

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