TY - CHAP
T1 - Measuring transmembrane helix interaction strengths in lipid bilayers using steric trapping
AU - Hong, Heedeok
AU - Chang, Yu-Chu
AU - Bowie, James U.
PY - 2013
Y1 - 2013
N2 - 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 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.
AB - 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 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.
KW - Biotinylation
KW - Glycophorin A dimer
KW - Membrane protein folding
KW - Monovalent streptavidin
KW - Steric trap
KW - Transmembrane helix interaction
UR - http://www.scopus.com/inward/record.url?scp=84884171561&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84884171561&partnerID=8YFLogxK
U2 - 10.1007/978-1-62703-583-5_3
DO - 10.1007/978-1-62703-583-5_3
M3 - Chapter
C2 - 23975771
AN - SCOPUS:84884171561
SN - 9781627035828
VL - 1063
T3 - Methods in Molecular Biology
SP - 37
EP - 56
BT - Methods in Molecular Biology
ER -