Nuclear Magnetic Resonance (NMR) of Proteins: Solid State

David A Middleton, University of Liverpool, Liverpool, UK

Published online: October 2012

DOI: 10.1002/9780470015902.a0003106.pub2

Solid-state nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for examining the structures of protein assemblies and complexes that are unsuitable for analysis by diffraction methods. Structural details are obtained for challenging systems including membrane proteins in lipid bilayers and water-insoluble fibrillar proteins. High-resolution spectra of proteins in solid or gel phases lacking long-range order are obtained with magic-angle spinning and, for membrane-embedded proteins, further anisotropic information is gained from spectra of stationary, macroscopically aligned samples. The information obtained defines the secondary structure and global fold of the protein, and also the orientation and topology of membrane proteins within lipid bilayers. These methods have elucidated, amongst other things, the structures of ion channels and their interactions with antiviral drugs and toxins and the molecular architectures of amyloid fibrils associated with Alzheimer's disease and type II diabetes. In addition, the molecular conformations of numerous ligands have been determined in the sites of action within their biological receptor proteins.

Key Concepts:

  • For solid-state NMR measurements on biological materials, samples are either macroscopically aligned or are rotated at the magic-angle.
  • Solid-state NMR measurements on aligned membrane proteins provide anisotropic restraints on the orientations of contiguous peptide planes or domains relative to the lipid bilayer.
  • Magic-angle spinning provides high-resolution spectra containing information on isotropic chemical shifts and interatomic distances and torsional angles.
  • Both approaches require isotopic labelling (13C and 15N supplemented as necessary with deuteration) of the protein.

Keywords: oriented samples; magic-angle spinning; membrane protein; ion channel; amyloid fibrils; ligand conformation; receptor; HIV; influenza

Figure 1. One-dimensional solid-state 15N NMR spectra of the three Vpu constructs, obtained at 0 °C in oriented lipid bilayers: (a) Vpu, (b) Vpu2–51 and (c) Vpu28–81. The orientations of transmembrane and in-plane amide N–H bonds are indicated above the spectra. The top row shows the overall architecture of the three constructs utilised in this study in the context of a membrane bilayer, with the hydrophobic transmembrane helix in red and the two amphipathic helices in blue (Marassi et al., 1999).
Figure 2. (a) Solid-state 1H–15N dipolar/15N chemical shift correlation PISEMA NMR spectrum of uniformly 15N-labelled nAChR M2 in oriented dimyristoylphosphatidylcholine bilayers at 22 °C. (b) Structure of the nAChR M2 funnel-like pentameric bundle calculated using the solid-state NMR coordinates of the M2 helix in the lipid bilayer, and by imposing a symmetric pentameric organisation. The wide mouth of the funnel is on the N-terminal, intracellular side of the pore. The C-terminus is on top (Opella et al., 1999). (c) Structure of the conductance domain of the M2 proton channel of influenza A in lipid bilayers. The tetramer is viewed down through the transmembrane channel from the virus interior. The amphipathic helices (the structural elements closest to the viewer) are oriented approximately perpendicular to the transmembrane helices. The residues represented as sticks form the His37-Trp41 tetrameric cluster involved in proton conduction.
Figure 3. Example of MAS solid-state NMR methods to examine the molecular architecture of an amyloid fibril (Madine et al., 2008). The fibrillar peptide is H2N-SNNFGAILSS-COOH. Chemical shift measurements (not shown) are consistent with the fibrillar peptide assuming an unbroken -strand conformation. (a) Measurements of intermolecular dipolar couplings between 13C backbone sites, using the dipolar recoupling method of rotational resonance, translate into an interatomic distance of 5.5 Å. (b) Such a short distance is possible if the peptide -strands are hydrogen bonded in a parallel, not antiparallel, arrangement (right). (c) A two-dimensional DARR NMR spectrum shows cross-peaks correlating intraresidue couplings within L27 (red) and an inter-residue coupling between the aromatic carbons of F23 at ~125 ppm and C of L27 (green), consistent with close contact between the phenylalanine and leucine residues. (d) The fibrils must consist of at least two sheets of parallel hydrogen-bonded -strands packed together with the sheets antiparallel to each other.
Figure 4. The molecular conformation of adenosine 5¢-triphosphate (ATP) in the nucleotide site of Na,K-ATPase as determined by REDOR solid-state NMR measurements of intramolecular 13C–31P distances. (a) The chemical structure of ATP showing the three structurally diagnostic distances measured: C8-P, C8-P and C8-P. (b) 31P REDOR spectra of ATP bound to Na,K-ATPase at –30 °C. The 13C–31P distances are proportional to the peak intensity reduction in the red spectrum compared to the spectrum in black. (c) The statistically favoured experimentally restrained ATP conformation modelled in the Na,K-ATPase nucleotide site (Middleton et al., 2011). Dashed lines represent polar contacts from ATP to residues known to be sensitive to nucleotide binding.
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Related Sub-Topics:

Membrane Proteins | Protein Structure | Nuclear Magnetic Resonance | Proteins: Structure, Function, Metabolism
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Article Title: Nuclear Magnetic Resonance (NMR) of Proteins: Solid State
 
How to Cite close
Middleton, David A(Oct 2012) Nuclear Magnetic Resonance (NMR) of Proteins: Solid State. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003106.pub2]