The Influence of Cholesterol on Fast Dynamics Inside of Vesicle and Planar Phospholipid Bilayers Measured with 2D IR Spectroscopy

Abstract

Phospholipid bilayers are frequently used as models for cell membranes. Here the influence of cholesterol on the structural dynamics in the interior of 1,2-dilauroyl-sn-glycero-3-phosphocholine (dilauroylphosphatidylcholine, DLPC) vesicles and DLPC planar bilayers are investigated as a function of cholesterol concentration. 2D IR vibrational echo spectroscopy was performed on the antisymmetric CO stretch of the vibrational probe molecule tungsten hexacarbonyl, which is located in the interior alkyl regions of the bilayers. The 2D IR experiments measure spectral diffusion, which is caused by the structural fluctuations of the bilayers. The 2D IR measurements show that the bilayer interior alkyl region dynamics occur on time scales ranging from a few picoseconds to many tens of picoseconds. These are the time scales of the bilayers' structural dynamics, which act as the dynamic solvent bath for chemical processes of membrane biomolecules. The results suggest that at least a significant fraction of the dynamics arise from density fluctuations. Samples are studied in which the cholesterol concentration is varied from 0% to 40% in both the vesicles (72 nm diameter) and fully hydrated planar bilayers in the form of aligned multibilayers. At all cholesterol concentrations, the structural dynamics are faster in the curved vesicle bilayers than in the planar bilayers. As the cholesterol concentration is increased, at a certain concentration there is a sudden change in the dynamics, that is, the dynamics abruptly slow down. However, this change occurs at a lower concentration in the vesicles (between 10% and 15% cholesterol) than in the planar bilayers (between 25% and 30% cholesterol). The sudden change in the dynamics, in addition to other IR observables, indicates a structural transition. However, the results show that the cholesterol concentration at which the transition occurs is influenced by the curvature of the bilayers.

Ultrafast Dynamics at Lipid–Water Interfaces

“…Interestingly, the dynamics of W­(CO) 6 are insensitive to the amount of hydration water present, suggesting that dynamics in the hydrophobic region are largely decoupled from the headgroup. Vesicles exhibit faster picosecond-scale dynamics compared to planar multibilayers, indicating that membrane curvature plays a role in lipid-packing and dynamics within the bilayer core . Metal carbonyls have also been covalently attached to cholesterol, and the measured dynamics were more than twice as slow for the same probe in bulk water .…”

“…In saturated lipids, the only feasible modes used to probe the bilayer core are the CH 2 or CH 3 stretching modes. Vibrational coupling makes it difficult to obtain an atomistic interpretation of measurements in this frequency region, and for that reason, extrinsic probes such as metal carbonyls have been introduced to measure dynamics within the hydrophobic core. , In past studies, the combination of vibrational probes with ultrafast spectroscopy has provided numerous insights into protein environments and dynamics, and the same methods can be directly translated to membrane studies . Probes become useful when the intrinsic vibrational modes are weak, insensitive to their environment, or when spectra are difficult to interpret due to spectral congestion or other factors.…”

“…Studying lipid diversity is an active area of research as simulation tools become better equipped for modeling heterogeneous, complex membranes , and as advances in lipidomics quantify the diversity of lipids in cell membranes. − Some of the recent ultrafast work has focused on the effects of increasing levels of heterogeneity and molecular diversity on model membranes. For example, cholesterol has been linked to domain formation, and consequently, recent efforts have focused on understanding the effect of cholesterol on bilayer dynamics. , …”

Applications of two-dimensional infrared spectroscopy

“…Large changes in structural dynamics occur upon bilayer phase transitions induced by dehydration or cholesterol addition, and furthermore, the changes are influenced by the degree of bilayer curvature. 51, 52, 120 The combination of similar probes with 2D IR spectroscopy could advance our understanding of membrane biophysics and potentially how variations in membrane properties due to lipid or protein composition might influence biological systems.…”

“…In addition to the associated water at the membrane interface, 2D IR spectroscopy can yield insight into interior membrane structure and dynamics. 51,52,113,120 The use of nonpolar molecules as vibrational probes permits selective measurement of the spectral dynamics due to fluctuations within the interior hydrophobic bilayer environment. 51,113 For example, recent experiments took advantage of the intense absorption bands of the small, nonpolar molecular probe tungsten hexacarbonyl to measure the dynamics of both planar and vesicular bilayers with 2D IR spectroscopy.…”