Lipid Transfer Kinetics from Nanolipoprotein Particles to Bicelles

Abstract

Nanolipoprotein particles (NLPs), also known as nanodiscs, are lipid bilayers bounded by apolipoprotein. We recently showed that 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. Because monomeric membrane proteins can be prepared in NLPs by cell-free protein synthesis, the bicelle-induced transfer process may provide a new method for studying membrane protein oligomerization. The mechanism of the NLP-bicelle interaction is unknown. We have now tested the effects of bicelle detergent (DHPC), apolipoprotein (MSP1E3D1), and temperature on lipid transfer from NLPs to bicelles, using stopped-flow kinetics. NLPs were prepared with fluorescent lipids (0.02 to 0.05 mole fraction), consisting of FRET donors (NBD-PE) and acceptors (LR-PE) at approximately equal concentrations. NLPs were mixed with a 200-fold molar excess of DHPC/DMPC bicelles (equimolar 6- and 14-carbon acyl chains) in a stopped-flow fluorometer. The rate of lipid transfer was monitored by the appearance of unquenched NBD fluorescence at 520 nm. The observed pseudo-first-order rate constant was surprisingly small (0.26/sec). 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 biphasic. The results suggest NLP-bicelle mixing kinetics may be mechanistically similar to lipid mixing via fusion pores.