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Solid-State NMR of Membrane Proteins in Phospholipid Bicelles

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Introduction

Multidimensional solid-state NMR is a powerful tool for structural studies of membrane proteins reconstituted into fully hydrated phospholipid bilayers. Our approach takes advantage of the simplifications that result from from spontaneous uniaxial orientation of phospholipid bidelles in a magnetic field [1-3]. It is shown here that one- and twodimensional solid-state NMR spectra of membrane proteins in perpendicular (i.e., "unflipped") bicelles, display resolved resonances in both ¹⁵N chemical shift and dipolar coupling dimensions, demonstrationg that this system can be used to obtain structural information.

Advantages of Bicelles

·Alternative model membrane system. Useful for protein with extra-membrane loops. ·Samples are easily prepared. ·Hydration. Easy to maintain in glass tubes (~70-80% w/v). ·Magnetic orientation. No glass in bulk sample volume. Solenoid-coil probes (more efficient).	Figure 1. NMR Samples: Bicelles and Bilayers	Figure 2. 700 MHz/62 mm solenoid-coil, double-tuned probe.
Most available lipids yield the "wrong" 90° orientation. Lanthanides may be used to "flip" bicelles [4], but they induce paramagnetic broadening of some resonances. Evidence of motional narrowing in perpendicular ( "unflipped") bicelles suggests that this system is suitable for structural studies.		Figure 2. 700 MHz/62 mm solenoid-coil, double-tuned probe.

Simulations

Transmembrane -helix in perpendicular bicelles (S_zz = -1/2):
Fig. 3 - 1. Static case 2. Rotational Diffusion

Figure 3. (top) Effect of fast uniaxial averaging on PISEMA spectra of perpendicular bicelles, i.e., magnetically oriented at 90°. The α-helix is tilted at 30° relative to the bicelle normal. The static case yields a reduced powder pattern.

Extra-membrane α-helix in perpendicular bicelles (S_zz = -1/2):
Figure 4. (right) Effect of fast uniaxial averaging on PISEMA spectra of perpendicular bicelles magnetically oriented. α-helix tilted at 90° relative to the bicelle normal.

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