Opportunities and Challenges of Backbone, Sidechain, and RDC Experiments to Study Membrane Protein Dynamics in a Detergent-Free Lipid Environment Using Solution State NMR

Abstract

Whereas solution state NMR provided a wealth of information on the dynamics landscape of soluble proteins, only few studies have investigated membrane protein dynamics in a detergent-free lipid environment. Recent developments of smaller nanodiscs and other lipid-scaffolding polymers, such as styrene maleic acid (SMA), however, open new and promising avenues to explore the function-dynamics relationship of membrane proteins as well as between membrane proteins and their surrounding lipid environment. Favorably sized lipid-bilayer nanodiscs, established membrane protein reconstitution protocols and sophisticated solution NMR relaxation methods probing dynamics over a wide range of timescales will eventually reveal unprecedented lipid-membrane protein interdependencies that allow us to explain things we have not been able to explain so far. In particular, methyl group dynamics resulting from CEST, CPMG, ZZ exchange, and RDC experiments are expected to provide new and surprising insights due to their proximity to lipids, their applicability in large 100+ kDa assemblies and their simple labeling due to the availability of commercial precursors. This review summarizes the recent developments of membrane protein dynamics with a special focus on membrane protein dynamics in lipid-bilayer nanodiscs. Opportunities and challenges of backbone, side chain and RDC dynamics applied to membrane proteins are discussed. Solution-state NMR and lipid nanodiscs bear great potential to change our molecular understanding of lipid-membrane protein interactions.

Lipid Nanodiscs for High-Resolution NMR Studies of Membrane Proteins

“…Polymer, saposin, and peptide nanodiscs are less rigid, but the size of the nanodisc particles is less homogeneous. ,,,, This is caused by a discontinuous saposin or peptide belt around the lipid nanodisc or by the polydispersity of the used polymers. Although the average size of these nanodiscs can be adjusted by changing the molar ratio of scaffold material and lipid molecules, the size control is less well understood than with MSP nanodiscs.…”

“…Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique to obtain structural and dynamical insights on biomolecules at atomic resolution. − In contrast to other structural techniques, solution-state NMR can probe structural and dynamical features of biomolecules in a native soluble form. − This also holds true for MPs, where the properties and fluidity of the membrane environment are important factors for functionality. ,,,, However, due to the inherent physical limitations of the method, structure determination by solution-state NMR is currently limited to an approximate size range of 40–50 kDa. − Nonetheless, it has been shown that solution-state NMR can be used to probe dynamics and conformational features of large (membrane) proteins up to 100 kDa to 1 MDa in size, utilizing specific methyl labeling of the proteins. , With such a strategy, NMR can be very efficiently used in a complementary manner in concert with X-ray crystallography and cryo-EM. The specialty of NMR is the structure determination of small and dynamic MPs, probing the dynamical features of MPs and monitoring the structural and dynamical effects of ligand binding.…”

“…251−253 This also holds true for MPs, where the properties and fluidity of the membrane environment are important factors for functionality. 46,54,63,241,254 However, due to the inherent physical limitations of the method, structure determination by solution-state NMR is currently limited to an approximate size range of 40−50 kDa. 255−259 Nonetheless, it has been shown that solution-state NMR can be used to probe dynamics and conformational features of large (membrane) proteins 241 up to 100 kDa to 1 MDa in size, utilizing specific methyl labeling of the proteins.…”

“…46,54,63,241,254 However, due to the inherent physical limitations of the method, structure determination by solution-state NMR is currently limited to an approximate size range of 40−50 kDa. 255−259 Nonetheless, it has been shown that solution-state NMR can be used to probe dynamics and conformational features of large (membrane) proteins 241 up to 100 kDa to 1 MDa in size, utilizing specific methyl labeling of the proteins. 260,261 With such a strategy, NMR can be very efficiently used in a complementary manner in concert with X-ray crystallography and cryo-EM.…”

Nanodiscs: A toolkit for membrane protein science

“…Nanodiscs have also been used for structural studies of membrane proteins in a detergent‐free lipid environment by NMR 45,119,120 . Although the large sizes of Nanodiscs often put limitations on high resolution NMR spectroscopy in solution, the advantages of this method of membrane protein solubilization can be used by applying new experimental tools to solve this problem 121 .…”