Bicelles exhibiting magnetic alignment for a broader range of temperatures: a solid-state NMR study

Bicelles are increasingly used as model membranes to suitably mimic the biological cell membrane for biophysical and biochemical studies by a variety of techniques including NMR and X-ray crystallography. Recent NMR studies have successfully utilized bicelles for atomic-resolution structural and dynamic studies of antimicrobial peptides, amyloid peptides, and membrane-bound proteins. Though bicelles composed with several different types of lipids and detergents have been reported, the NMR requirement of magnetic alignment of bicelles limits the temperature range in which they can be used and subsequently their composition. Because of this restriction, low-temperature experiments desirable for heat-sensitive membrane proteins have not been conducted because bicelles could not be aligned. In this study, we characterize the magnetic alignment of bicelles with various compositions for a broad range of temperatures using (31)P static NMR spectroscopy in search of temperature-resistant bicelles. Our systematic investigation identified a temperature range of magnetic alignment for bicelles composed of 4:1 DLPC:DHexPC, 4:1:0.2 DLPC:DHexPC:cholesterol, 4:1:0.13 DLPC:DHexPC:CTAB, 4:1:0.13:0.2 DLPC:DHexPC:CTAB:cholesterol, and 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate. The amount of cholesterol-3-sulfate used was based on mole percent and was varied in order to determine the optimal amount. Our results indicate that the presence of 75 wt % or more water is essential to achieve maximum magnetic alignment, while the presence of cholesterol and cholesterol-3-sulfate stabilizes the alignment at extreme temperatures and the positively charged CTAB avoids the mixing of bicelles. We believe that the use of magnetically aligned 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate bicelles at as low as -15 °C would pave avenues to study the structure, dynamics, and membrane orientation of heat-sensitive proteins such as cytochrome P450 and could also be useful to investigate protein-protein interactions in a membrane environment.