Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Tuesday Speaker Abstracts

FLEXIBILITY OF LARGER NANODISCS ALLOWS FOR MORE NATIVE-LIKE PHYSICAL PROPERTIES OF INCORPORATED LIPIDS Mark J Arcario 1 ; Vikram K Dalal 1 ; Wayland W Cheng 1 ; 1 Washington University in Saint Louis, Anesthesiology, St. Louis, MO, USA In the current structural revolution brought about by cryo-electron microscopy (cryo-EM), nanodiscs have become an indispensable tool in understanding how the membrane environment affects membrane protein structure. Recent studies, however, have described subtle structural differences to cryo-EM structural models depending on the nanodisc scaffold size and type. While it is well-known that nanodiscs alter certain physical properties of lipid bilayers, it has been hypothesized that these alterations are due to the edge effect introduced by the nanodisc scaffold and that large enough nanodiscs would recover more native-like properties, given that proportionally less lipids would be perturbed by the rim. With the advent of nanodisc circularization technology, which makes nanodiscs up to 100 nm thermally stable, we have been able to test this hypothesis. Using nanodiscs circularized with the SpyCatcher-SpyTag modification we have tested the packing behavior of pure lipids and lipid mixtures in nanodiscs ranging from 11 nm in size to 50 nm in size using the environmentally-sensitive fluorophore, Laurdan. When compared to a 100 nm liposome of the same lipid composition, we have found that nanodiscs under 25 nm demonstrate a more ordered bilayer, have a higher melting temperature, and have significantly lower cooperativity in the phase transition. Importantly, however, increases in nanodisc size monotonically mitigate these perturbations to the lipid properties. At 50 nm, we are able to recover native-like membrane properties in a nanodisc, supporting a long-standing hypothesis in the field. To begin understanding why larger nanodiscs are more native-like, we analyzed coarse-grained molecular dynamics (CGMD) simulations of 15, 30, and 50 nm circularized nanodiscs. These simulations demonstrate that, indeed, proportionally more lipids demonstrate unperturbed physical properties compared to smaller nanodiscs, including lipid tilt and lipid diffusion. Overall, these results provide a deeper understanding of the properties of lipids contained in nanodiscs, which will help in interpretation of cryo-EM structures as well as the fundamental nature of protein-lipid interactions.

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