Determining the orientation of uniaxially rotating membrane proteins using unoriented samples: a 2H, 13C, AND 15N solid-state NMR investigation of the dynamics and orientation of a transmembrane helical bundle

J Am Chem Soc. 2007 May 2;129(17):5719-29. doi: 10.1021/ja070305e. Epub 2007 Apr 7.

Abstract

Membrane protein orientation has traditionally been determined by NMR using mechanically or magnetically aligned samples. Here we show a new NMR approach that abolishes the need for preparing macroscopically aligned membranes. When the protein undergoes fast uniaxial rotation around the bilayer normal, the 0 degrees -frequency of the motionally averaged powder spectrum is identical to the frequency of the aligned protein whose alignment axis is along the magnetic field. Thus, one can use unoriented membranes to determine the orientation of the protein relative to the bilayer normal. We demonstrate this approach on the M2 transmembrane peptide (M2TMP) of influenza A virus, which is known to assemble into a proton-conducting tetrameric helical bundle. The fast uniaxial rotational diffusion of the M2TMP helical bundle around the membrane normal is characterized via 2H quadrupolar couplings, C-H and N-H dipolar couplings, 13C chemical shift anisotropies, and 1H T1rho relaxation times. We then show that 15N chemical shift anisotropy and N-H dipolar coupling measured on these powder samples can be analyzed to yield precise tilt angles and rotation angles of the helices. The data show that the tilt angle of the M2TMP helices depends on the membrane thickness to reduce the hydrophobic mismatch. Moreover, the orientation of a longer M2 peptide containing both the transmembrane domain and cytoplasmic residues is similar to the orientation of the transmembrane domain alone, suggesting that the transmembrane domain regulates the orientation of this protein and that structural information obtained from M2TMP may be extrapolated to the longer peptide. This powder-NMR approach for orientation determination is generally applicable and can be extended to larger membrane proteins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Algorithms
  • Anisotropy
  • Cytoplasm / chemistry
  • Electromagnetic Fields
  • Lipid Bilayers
  • Lipids / chemistry
  • Magnetic Resonance Spectroscopy
  • Membrane Proteins / chemistry*
  • Membranes, Artificial
  • Models, Molecular
  • Peptides / chemistry
  • Phosphatidylcholines / chemistry
  • Temperature

Substances

  • Lipid Bilayers
  • Lipids
  • Membrane Proteins
  • Membranes, Artificial
  • Peptides
  • Phosphatidylcholines
  • 1,2-linoleoylphosphatidylcholine
  • 1-palmitoyl-2-oleoylphosphatidylcholine