Structure of the transmembrane region of the M2 protein H+ channel by solid state NMR spectroscopy
Structure of the transmembrane region of the M2 protein H(+) channel.
Helix tilt of the M2 transmembrane peptide from influenza A virus: an intrinsic property.
This sequence occurs naturally in the Influenza A virus (Udorn/72 strain). The M2 transmembrane peptide was synthesized using solid phase peptide synthesis.
15N CHEMICAL SHIFT, 1H-15N DIPOLAR COUPLING FREQUENCIES WERE MEASURED FROM SOLID STATE NMR PISEMA EXPERIMENT
The lowest energy conformer with backbone and C beta atoms is deposited, preferred rotameric states of side chains were used during the backbone structure refinement but the side chain atoms were not included in the pdb file.
The refined M2-TMP monomer structure was obtained by a geometrical search using a search algorithm to obtain a minimum of the global penalty function that incorporates all the orientational restraints and the CHARMM empirical function. The orientational restraints imposed on the structure during refinement are 15 15N chemical shifts and 15 15N-1H dipolar couplings from PISEMA experiments. The observed chemical shifts are compared to calculated values from the molecular coordinates and the known tensor element magnitudes and assumed tensor orientations. The refinement was carried out in vacuo with the initial coordinates of an ideal a-helix structure (3.6 residues per turn) having a range of tilt and rotational orientations with respect to the bilayer spanning the values obtained from the PISA wheels.
Oriented samples of the peptide in hydrated lipid bilayers were prepared by first co-dissolving M2-TMP and dimyristoylphosphatidylcholine (DMPC) in trifluoroethanol (TFE). The solution was then spread onto approximately 60 glass plates. After vacuum drying to remove TFE, 2 microliters of sterile-filtered water was added to each plate, and the plates were then stacked into a glass tube and placed in a chamber containing a saturated solution of K2SO4 for hydration.
Oriented bilayers formed after equilibrating the sample in this chamber at 42C overnight. This sample container was then sealed with a microscope cover glass and epoxy to maintain sample hydration during the experiments.
INFLUENZA A VIRUS, MEMBRANE PROTEIN STRUCTURE, M2 PROTON CHANNEL, SOLID STATE NMR, MEMBRANE PROTEIN