Magnetically aligned phospholipid bilayers in weak magnetic fields: optimization, mechanism, and advantages for X-band EPR studies

Abstract

Our lab is developing a spin-labeled EPR spectroscopic technique complementary to solid-state NMR studies to study the structure, orientation, and dynamics of uniaxially aligned integral membrane proteins inserted into magnetically aligned discotic phospholipid bilayers, or bicelles. The focus of this study is to optimize and understand the mechanisms involved in the magnetic alignment process of bicelle disks in weak magnetic fields. Developing experimental conditions for optimized magnetic alignment of bicelles in low magnetic fields may prove useful to study the dynamics of membrane proteins and its interactions with lipids, drugs, steroids, signaling events, other proteins, etc. In weak magnetic fields, the magnetic alignment of Tm 3+-doped bicelle disks was thermodynamically and kinetically very sensitive to experimental conditions. Tm 3+-doped bicelles were magnetically aligned using the following optimized procedure: the temperature was slowly raised at a rate of 1.9 K/min from an initial temperature being between 298 and 307 K to a final temperature of 318 K in the presence of a static magnetic field of 6300 G. The spin probe 3β-doxyl-5α-cholestane (cholestane) was inserted into the bicelle disks and utilized to monitor bicelle alignment by analyzing the anisotropic hyperfine splitting for the corresponding EPR spectra. The phases of the bicelles were determined using solid-state 2H NMR spectroscopy and compared with the corresponding EPR spectra. Macroscopic alignment commenced in the liquid crystalline nematic phase (307 K), continued to increase upon slowly raising the temperature, and was well-aligned in the liquid crystalline lamellar smectic phase (318 K).