Effects of Cholesterol on Nanodisc Formation and Magnetic Alignment in DMPC and Glycyrrhizic Acid Systems Probed by ³¹ P and ¹⁴ N Solid-State NMR

Nanodiscs and bicelles are widely used as membrane mimetics for structural studies of membrane-associated systems. Studies have reported that their magnetic alignment behavior and phase stability are highly sensitive to composition and temperature. In this study, we systematically investigate the effects of cholesterol on bicelle formation and magnetic alignment in DMPC + 0.2 glycyrrhizic acid (GA) systems using a combined ³¹ P and ¹⁴ N solid-state NMR experimental and simulation-based approach. Temperature dependent ³¹ P NMR spectra reveal a clear transition from vesicle dominant to aligned bicelles/nanodsics phase, while ¹⁴ N quadrupolar splitting and lineshape analysis provides quantitative insights into heterogeneous lipid bilayer populations, distinguishing large aligned nanodiscs (B(L)), small nanodiscs (B(S)), and isotropic/random components (B(R)). A strong correlation is observed between the ³¹ P derived bicelle fraction and the ¹⁴ N B(L) population, confirming that macroscopic alignment in the presence of an external magnetic field directly reflects the growth of large, well-ordered nanodiscs. Cholesterol is found to play a critical dual role by modulating membrane order and curvature. At low cholesterol concentration (0-5 mol%), nanodiscs alignment occurs gradually with increasing temperature, while at higher cholesterol concentration (15–25 mol%), the alignment is delayed and accompanied by broader spectral features, indicating structural heterogeneity. Notably, 10 mol% cholesterol consistently provides the optimal balance, enabling efficient temperature dependent conversion to aligned bicelles while maintaining high B(L) populations (∼70-80%) and minimal isotropic fractions. In contrast, higher cholesterol maintains significant B(S) and B(R) populations, even at elevated temperature. The ¹⁴ N quadrupolar coupling (C _q ≈8.5–9.2 kHz for aligned nanodiscs) remains nearly invariant across compositions, showing that cholesterol does not change local headgroup dynamics but instead redistributes lipid populations. These findings establish a combined ³¹ P and ¹⁴ N solid -state NMR approach provides a valuable platform for quantitatively correlating membrane structure, dynamics, and alignment, offering practical guidelines for optimizing bicelle systems for high resolution solid-state NMR studies of membrane associated biomolecules.