(168s) Assay and Solid-State NMR Spectroscopy of Biomembranes and Soft Materials in a Hydrogel/Particle-Based Biomimetic Material System

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. At the project’s core, the overall materials science slant of the work is to fabricate a highly mechanically-stable particle/hydrogel-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 (~8x10⁶ x g) leads to the breakdown of the desired structure and thus the desired MP lipid microenvironment is compromised. The specific design hypothesis is that jammed particle systems altered with hydrogel networks can be used to form a mechanically-stable and continuous 3D supported biomembrane material that is externally accessible by lateral intramembrane diffusion. MAS-SSNMR is used to 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 is used in the validation of the biomembrane microenvironment and membrane protein concentration.

The specific design hypothesis is that jammed particle systems altered with hydrogel networks can be used to form a mechanically-stable and continuous 3D supported biomembrane materials. We have constructed systems composed with 1) 5 micron silica lipobeads: 2:2:1 (PC:SM:Chol (0.5 % DiO)) doped with 5 % phosphaditylcholine-PEG₂₀₀₀-NH-acrylate with in situ carboylmethycelulose polymerization and 2) 5 micron silica lipobeads DMPC/DiO doped with 5% diacetylene lipids/5 % PC-PEG₂₀₀₀-NH_-acrylate in situ carboylmethycelulose polymerization that are compared with untethered control proteolipobead systems. The hydrogels are doped with the fluorescent antenna protein phycocyanin to observe their structural integrity after MAS. We studied their structure in 3D using AIRYSCAN (confocal) superresolution microscopy and then their stability in magic-angle spinning solid state NMR (MAS-SSNMR) experiments going from low speed (5 Hz) to higher speeds (>50 kHz) at ambient temperatures We determined the threshold at which MAS speed the systems break down by monitoring the changes in residual ¹H₂O using proton MAS-NMR. To further examine the lipid structures we have incorporated intrinsically 19F labeled BODIPY-PC (green fluorescenent), enabling 19F-SS-NMR.