Nanolipoprotein particles (NLPs), also known as nanodiscs, are lipid bilayers bounded by apolipoprotein. Lipids and membrane proteins cannot exchange between NLPs. However, addition of bicelles opens NLPs and transfers their contents to bicelles, which freely exchange lipids and proteins. NLP-bicelle interactions may provide a new method for studying membrane protein oligomerization. The interaction mechanism was investigated by stopped flow fluorometry. NLP lipids included fluorescence resonance energy transfer donors and acceptors. NLPs were mixed with a 200-fold molar excess of dihexanoyl phosphatidylcholine (DHPC)/dimyristoyl phosphatidylcholine bicelles, and the rate of lipid transfer was monitored by the appearance of dequenched lipid donor fluorescence. The observed pseudo-first-order rate constant was surprisingly small. NLPs did not react with DHPC alone below its critical micelle concentration (cmc). Above the cmc, the reaction was complete within the instrument dead time. Thus, the rate-limiting step is not the reaction of NLPs with DHPC monomers or micelles. Added MSP1E3D1 had no effect on the rate, ruling out free apolipoprotein involvement. The NLP-bicelle mixing rate showed a strong temperature dependence (activation energy = 28 kcal/mol). Near or below the DMPC phase transition temperature, the kinetics were sigmoidal. Models are proposed for the NLP-bicelle mixing, including one involving fusion pores.

A new method for the measurement of membrane protein oligomer association is described. Two engineered fragments of bacteriorhodopsin, which are known to spontaneously associate in bicelles, were expressed in nanolipoprotein particles (NLPs or nanodiscs) using an Escherichia coli S30 cell-free synthesis system. When separately prepared NLPs containing the fragments were mixed, fragment association did not occur, indicating that the apolipoprotein edge blocks transfer between NLPs. However, when bicelles were added to this mixture, fragment association was detected by disulfide cross-linking. The rate of cross-linking was consistent with previously published equilibrium and kinetic parameters. Characterization of the NLP/bicelle mixture by dynamic light scattering and fluorescence spectroscopy indicates that the NLP bilayer transfers to bicelles in a simple reversal of the synthesis of NLPs from bicelles. These experiments validate using cell-free synthesis of membrane proteins in NLPs, followed by treatment with bicelles, as a method for measuring oligomerization of integral membrane protein subunits in a bilayer-like environment.

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