(55b) A Particle-Based Network Biomimetic Material System for Assay and Spectroscopy of Membrane Proteins in Intact Lipid Bilayers

The objective of this work is to develop a supported biomembrane network material platform for solid-state NMR spectroscopy and assay of membrane proteins in intact lipid bilayers. This biomimetic material constructed in this platform enables NMR studies not currently possible and most importantly, allows for control and the spectroscopic validation of lipid microenvironment and the embedded membrane protein (MP) concentration. At the project’s core, the overall materials science slant of the work is to fabricate a highly mechanically-stable particle-based 3D network than can enable magic-angle spinning solid state NMR (MAS-SSNMR) at high speeds at ambient temperatures, while maintaining the biomembrane microenvironment. This is a current limitation in the field, as when attempting to achieve the desired resolution enhancement afforded by spinning at MAS frequencies of 40 kHz and higher, immense centrifugal forces acting on unsupported soft lipid bilayers (~10⁶ x g) leads to the breakdown of the desired structure and thus the desired MP lipid microenvironment is compromised. Additionally, this material is based on the formation of a continuous 3D network of laterally–mobile tether-supported biomembranes. This feature enables loading of membrane proteins by lateral intramembrane diffusion into the sample biomembrane region from “source” biomembranes brought into contact and subsequently fused for loading. The implications of implementing this new mode of membrane protein loading is an unprecedented advance that we expect will ultimately allow for the staging of complex protein-protein interactions in the assay/spectroscopy sample volume.

The specific design hypothesis is that jammed particle systems can be used to form a mechanically-stable and continuous 3D supported biomembrane material that is externally accessible by lateral intramembrane diffusion. The goal of this overall research direction is to build systems that could be used to detect and characterize how lipid microenvironment affects the enzyme and its substrate. MAS-SSNMR was used in tandem to monitor and validate MP and lipid concentrations in the continuous network of tether-supported biomembranes contained in the assay volume. In tandem with SSNMR, super-resolution 3D imaging microscopy was used in the validation of the biomembrane microenvironment and membrane protein concentration.