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. 2012 Jul 3;103(1):29-37.
doi: 10.1016/j.bpj.2012.05.016.

Supramolecular Structure of Membrane-Associated Polypeptides by Combining Solid-State NMR and Molecular Dynamics Simulations

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Supramolecular Structure of Membrane-Associated Polypeptides by Combining Solid-State NMR and Molecular Dynamics Simulations

Markus Weingarth et al. Biophys J. .
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Abstract

Elemental biological functions such as molecular signal transduction are determined by the dynamic interplay between polypeptides and the membrane environment. Determining such supramolecular arrangements poses a significant challenge for classical structural biology methods. We introduce an iterative approach that combines magic-angle spinning solid-state NMR spectroscopy and atomistic molecular dynamics simulations for the determination of the structure and topology of membrane-bound systems with a resolution and level of accuracy difficult to obtain by either method alone. Our study focuses on the Shaker B ball peptide that is representative for rapid N-type inactivating domains of voltage-gated K(+) channels, associated with negatively charged lipid bilayers.

Figures

Figure 1
Figure 1
(a) Sequence of the ShB peptide. Residues with defined structure according to ssNMR are indicated in bold. (b) SPECIFIC NCOCX (49) (red) and NCA (blue) 15N-13C correlation spectra as well as PDSD 13C-13C correlation spectra obtained under weak C′-Cα recoupling (50) (green) showing intraresidual (black), sequential (blue), and interstrand (red) connectivity. (c) NHHC (51) spectrum recorded with a proton mixing time of 150 μs. Intraresidue correlations are labeled black, sequential correlations blue. (d) Cα region of a CHHC (51) spectrum employing 500 μs proton mixing. Intraresidue crosspeaks are labeled and expected sequential crosspeaks are marked by blue crosses. Red crosses indicate long-range correlations expected for the β-hairpin shown in e: Potential β-hairpin of the ShB peptide. Expected long-range Cα-Cα and, accordingly, Hα-Hα contacts are indicated by red lines. (f) Spectrum of a PARIS-xy (52) 13C-13C correlation experiment prompted by MD simulations. See online version for colored figure versions.
Figure 2
Figure 2
Comparison of (a and b) the ssNMR structure with (c and d) the ssNMR-MD structure. For the latter, eight new restraints were added (Table S6). The three lowest energy structures are superimposed. The inter-β-strand hydrogen bonds are indicated as green dashed lines.
Figure 3
Figure 3
Membrane topology of the ShB peptide obtained by ssNMR. (a) T2 filtered HHC spectrum obtained for a proton mixing time of 2 ms. Peptide resonances are indicated and lipid-protein and water-protein crosspeaks are colored brown and blue, respectively. Cross sections of lipid-peptide and water-peptide crosspeaks are shown in Fig. S15. (b) Integrals of water-protein (blue) crosspeaks computed from T2 filtered HHC spectra as a function of proton mixing time. (c) SsNMR topology of the ShB peptide. Phosphates are represented by spheres. See online version for colored figure versions.
Figure 4
Figure 4
Comparison of water access obtained with T2-edited HHC experiments (dark blue columns) and back-calculated from MD simulations (light blue columns) for some resolved peptide Cα-positions. In a, the water access was back-calculated for the peptide structure and topology obtained by ssNMR-MD and in b for the (frozen) peptide structure and topology obtained by ssNMR alone. The number of water molecules was averaged within a radius of 9 Å from (a) 30–330 and (b) 5–10 ns MD trajectories. (c) SsNMR-MD and (d) ssNMR structure and topology. Water molecules within a radius of 6 Å around the Cα of A3-G9 are shown for clarity. See online version for colored figure versions.
Figure 5
Figure 5
Supramolecular structure of membrane-associated ShB peptide. The lipid bilayer surface is shown in gray, the peptide in red cartoon. Water molecules around peptide residues 1-12 within a radius of 9 Å are illustrated in blue. Hydrogen bonds between the β-strands and peptide-lipid hydrogen bonds are shown in green and orange, respectively. Distorted lipids (in gray lines) enable the formation of lipid-peptide hydrogen bonds. Interaction of the positively charged peptide tail with the negatively charged bilayer is illustrated. The snapshot was taken after 300 ns of the free simulation. See online version for colored figure versions.

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