Dataset Information

Immobilization of the influenza A M2 transmembrane peptide in virus envelope-mimetic lipid membranes: a solid-state NMR investigation.

ABSTRACT: The dynamic and structural properties of membrane proteins are intimately affected by the lipid bilayer. One property of membrane proteins is uniaxial rotational diffusion, which depends on the membrane viscosity and thickness. This rotational diffusion is readily manifested in solid-state NMR spectra as characteristic line shapes and temperature-dependent line narrowing or broadening. We show here that this whole-body uniaxial diffusion is suppressed in lipid bilayers mimicking the composition of eukaryotic cell membranes, which are rich in cholesterol and sphingomyelin. We demonstrate this membrane-induced immobilization on the transmembrane peptide of the influenza A M2 (AM2-TM) proton channel protein. At physiological temperature, AM2-TM undergoes uniaxial diffusion faster than approximately 10(5) s(-1) in DLPC, DMPC, and POPC bilayers, but the motion is slowed by 2 orders of magnitude, to <10(3) s(-1), in a cholesterol-rich virus envelope-mimetic membrane ("viral membrane"). The immobilization is manifested as near rigid-limit (2)H quadrupolar couplings and (13)C-(1)H, (15)N-(1)H, and (13)C-(15)N dipolar couplings for all labeled residues. The immobilization suppresses intermediate time scale broadening of the NMR spectra, thus allowing high-sensitivity and high-resolution spectra to be measured at physiological temperature. The conformation of the protein in the viral membrane is more homogeneous than in model PC membranes, as evidenced by the narrow (15)N lines. The immobilization of the M2 helical bundle by the membrane composition change indicates the importance of studying membrane proteins in environments as native as possible. It also suggests that eukaryote-mimetic lipid membranes may greatly facilitate structure determination of membrane proteins by solid-state NMR.

SUBMITTER: Luo W

PROVIDER: S-EPMC4082982 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Immobilization of the influenza A M2 transmembrane peptide in virus envelope-mimetic lipid membranes: a solid-state NMR investigation.

Luo Wenbin W Cady Sarah D SD Hong Mei M

Biochemistry 20090701 27

The dynamic and structural properties of membrane proteins are intimately affected by the lipid bilayer. One property of membrane proteins is uniaxial rotational diffusion, which depends on the membrane viscosity and thickness. This rotational diffusion is readily manifested in solid-state NMR spectra as characteristic line shapes and temperature-dependent line narrowing or broadening. We show here that this whole-body uniaxial diffusion is suppressed in lipid bilayers mimicking the composition ...[more]

PMID: 19489611

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Project description:The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the side-chain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting structural findings from non-bilayer samples.

Project description:The use of side chain methyl (13)C chemical shifts for the determination of the rotameric conformation of Val and Leu residues in proteins by solid-state NMR spectroscopy is described. Examination of the solution NMR stereospecifically assigned methyl groups shows significant correlation between the difference in the two methyl carbons' chemical shifts and the side chain conformation. It is found that alpha-helical and beta-sheet backbones cause different side chain methyl chemical shift trends. In alpha-helical Leu's, a relatively large absolute methyl (13)C shift difference of 2.89 ppm is found for the most populated mt rotamer (chi(1) = -60 degrees, chi(2) = 180 degrees), while a much smaller value of 0.73 ppm is found for the next populated tp rotamer (chi(1) = 180 degrees, chi(2) = 60 degrees). For alpha-helical Val residues, the dominant t rotamer (chi(1) = 180 degrees) has more downfield Cgamma2 chemical shifts than Cgamma1 by 1.71 ppm, while the next populated m rotamer (chi(1) = -60 degrees) shows the opposite trend of more downfield Cgamma1 chemical shift by 1.23 ppm. These significantly different methyl (13)C chemical shifts exist despite the likelihood of partial rotameric averaging at ambient temperature. We show that these conformation-dependent methyl (13)C chemical shifts can be utilized for side chain structure determination once the methyl (13)C resonances are accurately measured by double-quantum (DQ) filtered 2D correlation experiments, most notably the dipolar DQ to single-quantum (SQ) correlation technique. The advantage of the DQ-SQ correlation experiment over simple 2D SQ-SQ correlation experiments is demonstrated on the transmembrane peptide of the influenza A M2 proton channel. The methyl chemical shifts led to predictions of the side chain rotameric states for several Val and Leu residues in this tetrameric helical bundle. The predicted Val rotamers were further verified by dipolar correlation experiments that directly measure the chi(1) torsion angles. It was found that the chemical-shift-predicted side chain conformations are fully consistent with the direct torsion angle results; moreover, the methyl (13)C chemical shifts are sensitive to approximately 5 degrees changes in the chi(1) torsion angle due to drug binding.

Dataset Information

Immobilization of the influenza A M2 transmembrane peptide in virus envelope-mimetic lipid membranes: a solid-state NMR investigation.

Publications

Immobilization of the influenza A M2 transmembrane peptide in virus envelope-mimetic lipid membranes: a solid-state NMR investigation.

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