(294f) A Hydrogel/Particle-Based Biomimetic Material System for Assay and Solid-State NMR Spectroscopy of Biomembranes and Soft Materials

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/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 material. 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. 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 ¹H₂O using proton MAS-NMR. We concluded that higher T_m lipid compositions and those with polymerized lipids gain considerable stability enhancement over untethered low T_m lipid compositions.

Crossing the current soft material MAS barrier could enable high resolution NMR experiments not yet possible that could shed light on subpopulations of lipid and membrane protein resonances currently unresolvable and/or undetectable. This would lead to new studies that could uncover structural details about lipid-lipid and lipid-protein interactions that are currently obscured by insufficient resolution, low SNR or line broadening. If the inherent resolution can be enhanced, the field would be changed by opening up an expanded window of observation that could be used to elucidate new details about lipid microenvironments, phase separation and the role and influence of the fluid and complex lipid bilayer on the function of membrane proteins of biomedical relevance.