Solid state NMR: The essential technology for helical membrane protein structural characterization

Abstract

NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed - neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (Anfinsen, 1973) [106]. The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins.

Keywords: Helical membrane proteins; Magic angle spinning NMR; Membrane influence on structure; Membrane protein environment; Membrane protein structure; Oriented sample NMR; PISEMA; Solid state NMR; Structural validation.

Fig. 1

Crystal structures of (a) the tetrameric KcsA (PDB: 1BL8) [105] and the monomers of (b) KvAP (1ORQ) [1] and (c) Kv1.2 (PDB: 3LUT) [5] to simplify the view of these large structures. The conductance domain is in gray, the voltage sensing domain of KvAP and Kv1.2 with 4 TM helices is in green. The hydrophilic interfacial regions are pale blue bands underlying the three structures. The electron density associated with partial occupancy of the K⁺ ions is shown in purple. These ions and the conductance domain provide an accurate picture of the bilayer normal for orienting these structures.

Fig. 2

The conductance domain of the M2 proton channel from Influenza A shown in a lipid bilayer of dioleoylphosphatidylcholine and dioleoylphoshatidylethanolamine in which the structural data were obtained and in which the structure was refined using restrained molecular dynamics (PDB: 2L0J) [80]. The protein structure shown in yellow with a helical cartoon and sticks for the heavy atoms is a tetrameric structure composed of a TM and an amphipathic helix, the latter interacting with both the hydrophobic and hydrophilic regions of the bilayer. The heavy atoms of the lipids are shown as space filling spheres. Carbon is green or yellow, oxygen is red, phosphorous is orange, and nitrogen is blue.

Fig. 3

Dimeric gramicidin A structures viewed perpendicular (top) and parallel (bottom) to the pore axis (bottom). (a and b) The ‘head to head’ single stranded structure known to be a monovalent cation selective channel in membranes (PDB: 1MAG) [55]; (c and d) double helical antiparallel structure, not observed in lipid bilayers (PDB: 2XDC) [45]; and (e and f) another double helical antiparallel structure this time Cs⁺ bound in the pore, but again, not observed in lipid bilayers (PDB: 1GMK) [69].

Fig. 4

Structures of the M2 proton channel from Influenza A viewed perpendicular (top) and parallel (bottom) to the pore axis (bottom). The hydrophilic interfacial regions are pale yellow bands underlying the three structures and in between is the hydrophobic region of the bilayer. (a and b) An asymmetric X-ray crystal structure (PDB: 3BKD) [82] of the TM domain (residues 22–46) obtained from detergent based crystals; (c and d) a solution NMR structure of the conductance domain obtained from DHPC micelles (residues 18–60, PDB: 2RLF) [83] with bound rimantadine on the lipid facing surface (space filling view); (e and f) a ssNMR structure of the conductance domain (residues 22–62, PDB: 2L0J) [80] from liquid crystalline bilayers of DOPC and DOPE. The His37 residues shown in space filling view.

Fig. 5

Comparison of a sample of observed and predicted OS ssNMR data from wild type constructs of the Influenza A M2 protein. (Red) Observed resonance frequencies for S31, H37, W41, L43, L46, F47, F55, and L59 from M2 (22–62) in liquid crystalline lipid bilayers used to define the 2L0J structure [80]. The letter designations are used only for the 2L0J resonances. (Purple) Predicted resonance frequencies from the solution NMR structure (2RLF) [83] in detergent micelles of M2 (19–61) for the same sites. (Green) Predicted resonance frequencies from the X-ray crystal structure (3BKD) [82] in detergent based crystals of M2 (22–46) for the same sites except for F47, F55, and L59 which were not in this construct. 3BKD is an asymmetric structure and hence the four helices give rise to different resonance frequencies. Different shades of green are used to color code the different helices in 3BKD. The data and predictions are plotted on an absolute scale for the dipolar coupling. Both the predications and data for F47, F55, and L59 are used in the structures as negative values.