Measuring the energetics of membrane protein dimerization in mammalian membranes.
Thus far, quantitative studies of lateral protein interactions in membranes have been restricted peptides or simplified protein constructs in lipid vesicles or bacterial membranes. Here we show how free energies of membrane protein dimerization can be measured in mammalian plasma membrane-derived vesicles. The measurements, performed in single vesicles, utilize the quantitative imaging FRET (QI-FRET) method. The experiments are described in a step-by-step protocol. The protein characterized is the transmembrane domain of glycophorin A, the most extensively studied membrane protein, known to form homodimers in hydrophobic environments. The results suggest that molecular crowding in cellular membranes has a dramatic effect on the strength of membrane protein interactions.
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Lirong
L
Chen
Lawrence
L
Novicky
Mikhail
M
Merzlyakov
Tihomir
T
Hristov
Kalina
K
Hristova
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006021:Glycophorins / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014162:Transfection
D006021:Glycophorins; D008565:Membrane Proteins