Cholesterol Hydrogen Bonding Interactions in Lipid Membranes: Insights from Raman Fingerprint Modes*

While Raman spectroscopy has long provided insights into lipid membrane structure, progress has been largely limited to the vibrations of common functional groups within lipids. The more unique "fingerprint" vibrations of the entire molecule can be identified by comparisons to reference measurements, but a lack of detailed knowledge of their motion impairs their spectral interpretation. Here we will show that time-dependent density functional theory (TDDFT) can calculate accurate Raman spectra for molecules up to 100 atoms, including the effects of conformational variation and hydrogen bonding. A comparison between the calculated spectrum of cholesterol and measurements on purified cholesterol powder will be presented, identifying 34 vibrational modes. Furthermore, Raman peaks of cholesterol that are sensitive to hydrogen bonds at the hydroxyl group will be identified by TDDFT and confirmed with comparisons to powder and lipid vesicle spectra. Similar results on anthraquinones, flavonoids, and carotenoids will be presented. Together these results illustrate that TDDFT calculations create new opportunities for spectral interpretation in Raman spectroscopy and advance its applications in molecular structure analysis for biomolecules in their natural environment.