Abstract
Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein–protein interaction and oligomerization in a constraining environment.
Highlights
Interactions of integral membrane proteins trigger a variety of processes in cellular membranes making important contributions to cell function and having a high pharmacological importance [1]
In the present work we focus on lipid and protein dynamics in DIBMALPs of different size prepared at different protein-to-lipid ratios
At all selected diisobutylene/maleic acid (DIBMA)/lipid ratios, we could successfully obtain lipid nanoparticles.Dynamic light scattering (DLS) was employed to analyze the size distribution of particles in the preparations obtained upon lipid solubilization by the DIBMA copolymer
Summary
Interactions of integral membrane proteins trigger a variety of processes in cellular membranes making important contributions to cell function and having a high pharmacological importance [1]. Membrane proteins enclosed in stable polymer-stabilized lipid particles can be studied by different biophysical methods without loss of their structural and functional properties [13,26,27]. We apply EPR spectroscopy to study possible effects of varying sizes of DIBMALPs on the dynamics of enclosed lipids in the absence and presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII). To this end, we use a set of phosphatidylcholine lipids with nitroxide groups located at the 5th, 12th or 16th carbon atom positions along the hydrocarbon chain of the lipid and the saturated neutral lipid 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). CG MD simulations complement our experiments and show formation of stable DIBMALPs with an overall size up to 35 nm and uniform dynamics of the enclosed lipids
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