Premium
Formation of integral membrane protein oligomers
Author(s) -
Lai Yi-chun,
Renthal Robert
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.602.2
Subject(s) - homomeric , chemistry , oligomer , bacteriorhodopsin , model lipid bilayer , transmembrane domain , transmembrane protein , biophysics , lipid anchored protein , membrane protein , helix (gastropod) , crystallography , monomer , lipid bilayer , membrane , protein subunit , biochemistry , biology , receptor , polymer , polymer chemistry , apoptosis , ecology , organic chemistry , autophagy , snail , gene , lipid bilayer phase behavior
Many transmembrane (TM) proteins form oligomeric structures involving strong homomeric or heteromeric associations with other TM protein subunits. Little is known about the assembly mechanisms of these oligomers. We now report a technique for measuring the association of TM proteins in vitro . Bacteriorhodopsin fragments (2 and 5 TM helices) that are known to associate in detergent/lipid bicelles and form a native structure were separately inserted into nanolipoprotein particles (NLPs) using an E. coli cell‐free expression system. Each fragment contained one engineered cysteine, positioned so that disulfides form when the fragments correctly associate. The fragment/NLP complexes were mixed and disulfide crosslinking was initiated by Cu(II). No crosslinks between 2 and 5 TM helix fragments were detected. However, if the NLP complexes were mixed with bicelles prior to Cu(II) treatment, 2 to 5 TM helix crosslinks were observed. Thus, detergent/lipid bicelles open NLPs and permit protein mixing. The association constant for oligomer formation can be determined from the rate of crosslinking at varying protein concentrations. Future applications of this method include measuring association constants of protomers that form both homo‐ and hetero‐oligomeric channels (e.g. connexins, ligand‐gated ion channels), and studies of interactions of monomers with membrane chaperones.