Lipid Internal Dynamics Probed in Nanodiscs

Abstract

Nanodiscs offer a very promising tool to incorporate membrane proteins into native‐like lipid bilayers and an alternative to liposomes to maintain protein functions and protein–lipid interactions in a soluble nanoscale object. The activity of the incorporated membrane protein appears to be correlated to its dynamics in the lipid bilayer and by protein–lipid interactions. These two parameters depend on the lipid internal dynamics surrounded by the lipid‐encircling discoidal scaffold protein that might differ from more unrestricted lipid bilayers observed in vesicles or cellular extracts. A solid‐state NMR spectroscopy investigation of lipid internal dynamics and thermotropism in nanodiscs is reported. The gel‐to‐fluid phase transition is almost abolished for nanodiscs, which maintain lipid fluid properties for a large temperature range. The addition of cholesterol allows fine‐tuning of the internal bilayer dynamics by increasing chain ordering. Increased site‐specific order parameters along the acyl chain reflect a higher internal ordering in nanodiscs compared with liposomes at room temperature; this is induced by the scaffold protein, which restricts lipid diffusion in the nanodisc area.

Structure and dynamics of a nanodisc by integrating NMR, SAXS and SANS experiments with molecular dynamics simulations

“…1). These experiments were performed at 10 • C, and based on results from previous NMR experiments on nanodiscs (Martinez et al, 2017) as well as a melting temperature ≈ 24 • C for DMPC, we expect the lipids to be in the gel-phase. Our SAXS and SANS data all exhibit a flat Guinier region at low and indicate no signs of aggregation ( Fig.…”

“…We continued to investigate the impact of temperature and the presence of the His-tags on both the SAXS measurements and the resulting geometrical model of ΔH5-DMPC. We acquired standard solution SAXS data for a new preparation of the ΔH5-DMPC nanodiscs, this time without His-tags and measured at both 10 • C and 30 • C. At these two temperatures the DMPC is expected to be dominantly in the gel and liquid phase, respectively, as they are below and above the melting transition temperature (Martinez et al, 2017) (see also DSC analysis below). We used a standard solution SAXS setup for these measurements, as this at present provides a better control of both the sample temperature and sample concentration than in the SEC-SAXS based measurement.…”

“…A high resolution structure of the MSP protein belt encircling the nanodisc was recently obtained from the small, helix-5-deleted nanodisc, MSP1D1ΔH5 (henceforth ΔH5), reconstituted with DMPC lipids (ΔH5-DMPC) (Bibow et al, 2017) by combining nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and transmission electron microscopy (TEM) (Bibow et al, 2017). While these experiments were performed on lipid-loaded nanodiscs, the study focused on the protein components, and on determining a time-and ensemble averaged structure of these, but left open the question of the role of the lipids (Martinez et al, 2017) as well as any structural dynamics of the overall nanodisc. Intriguingly, the resulting structure of the belt proteins corresponded to that of an almost circularly-shaped disc, while our previous SAXS/SANS investigations are clearly consistent with discs with an on-average elliptical cross-section (Skar-Gislinge et al, 2010;Midtgaard et al, 2015).…”

A guide to quantifying membrane protein dynamics in lipids and other native‐like environments by solution‐state NMR spectroscopy

“…2A), [58]. However, their partially denaturing properties, high degree of curvature and unnatural lateral pressure profile may introduce perturbances of MP structure, function and dynamics [45,[59][60][61][62]. Therefore, alternative membrane mimetics have been developed to embed the protein in a flat lipid bilayer, such as bicelles, which are composed of a detergent/lipid pair [63].…”

Does human P-glycoprotein efflux involve transmembrane alpha helix breakage?

“…Nanodiscs are derived from apolipoprotein A1, also called membrane scaffolding protein (MSP), an amphipathic protein in which the hydrophobic helices interact with the acyl chains of the lipids while the hydrophilic sides are exposed to the solvent, thus allowing the formation of a lipid bilayer patch in solution. However, it was recently reported that the nanodisc lipid internal dynamics and thermotropism may be substantially altered, abolishing gel-fluid phase transitions and increasing site-specific order parameters that can only be fine-tuned with the addition of cholesterol [15]. As it is known that P-gp reshapes the surrounding lipid environment up to a radius of 15-20 nm [16], and as the inferred nanodisc size is ~10 nm (containing 120-160 lipids) [15], under the temperature conditions required by cryo-EM and using relatively small nanodiscs, the protein conformational space may be perturbed.…”