Kinetic Mechanism of Lipidprotein Nanodisc Dissociation in Bicelles

Abstract

Nanodisc soluble lipid bilayer systems are vital in rendering membrane proteins soluble in aqueous solutions where they remain monodisperse and active in a membrane‐like environment. Nanodiscs or nanolipidprotein particles (NLPs), are bordered by an amphipathic helical protein belt or membrane scaffolding protein (MSP) mimicking a cell membrane. Past works have shown that fragments of bacteriorhodopsin when placed in separate nanodisc do not interact to form the functional protein. The introduction of bicelles enabled the fragments to form oligomers inside the bicelles. An analysis of the kinetics of the aforementioned NLP‐bicelle interaction showed a sigmoid curve suggesting the possibility of an autocatalyzed reaction. We hypothesized that the kinetics where due to MSP1E3D1′s dissociation from the nanodisc which catalyzed the reaction. Using fluorescently labeled lipids 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(lissamine rhodamine B sulfonyl), 1,2‐diphytanoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl) LR‐PE and NBD‐PE, respectively with 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) and MSP1E3D1 we constructed NLPs. Bicelles were prepared using 1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (DHPC) and DMPC. Kinetics of NLP‐bicelle interactions and fluorescently labeled lipid dispersion were observed measuring fluorescence resonance energy transfer (FRET) using stopped‐flow spectroscopy. The NLP‐bicelle interaction followed previously observed kinetics and continued to do so after the addition of excess MSP1E3D1. Our results show that MSP1E3D1 is not responsible for the observed kinetics of the transfer of lipids between NLPs and bicelles.