Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins.
Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep membrane proteins (MPs) water-soluble while stabilizing them biochemically. We have examined the factors that determine the size and dispersity of MP/APol complexes and studied the dynamics of the association, taking as a model system the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) trapped by A8-35, a polyacrylate-based APol. Molecular sieving indicates that the solution properties of the APol largely determine those of tOmpA/APol complexes. Achieving monodispersity depends on using amphipols that themselves form monodisperse particles, on working in neutral or basic solutions, and on the presence of free APols. In order to investigate the role of the latter, a fluorescently labeled version of A8-35 has been synthesized. Förster resonance energy transfer measurements show that extensive dilution of tOmpA/A8-35 particles into an APol-free medium does not entail any detectable desorption of A8-35, even after extended periods of time (hours-days). The fluorescent APol, on the other hand, readily exchanges for other surfactants, be they detergent or unlabeled APol. These findings are discussed in the contexts of sample optimization for MP solution studies and of APol-mediated MP functionalization.
Zoonens
Manuela
M
Laboratoire de Physico-Chimie Moléculaire des Membranes Biologiques, UMR 7099, Centre National de la Recherche Scientifique, and Institut de Biologie Physico-Chimique, Université Paris-7, 13 rue Pierre et Marie Curie, F-75005 Paris, France.
Giusti
Fabrice
F
Zito
Francesca
F
Popot
Jean-Luc
JL
D001393:Azoles; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D003902:Detergents; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009578:Nitrobenzenes; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011437:Propylamines / Q000138:chemical synthesis / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
C510797:7-nitrobenz-2-oxa-1,3-diazol-4-yl; D001393:Azoles; D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D009578:Nitrobenzenes; D011108:Polymers; D011437:Propylamines; D012996:Solutions; C499017:amphipol A8-35