Structure determination of membrane proteins by NMR spectroscopy

Abstract

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.

Fig. 1

Representations of proteins in mice lies (top), bicelles (middle), and bilayers (bottom) (Opella 1997).

Fig. 2

Principles of PISA wheels (Marassi and Opella 2000). (A) Helical wheel showing the 100° arc between adjacent residues that is a consequence of the periodicity of 3.6 residues per turn in an α-helix; (B) orientations of the principal elements of the spin interaction tensors associated with ¹⁵N in a peptide bond; (C) PISA wheel for an ideal α-helix; (D) dipolar wave for an ideal α-helix.

Fig. 3

Experimental data and PISA wheel and dipolar wave for the AchR. M2 ion channel peptide. (A) Helical wheel; (B) experimental PISEMA spectrum; (C) PISA wheel for an ideal α-helix with a 12° tilt; (D) dipolar wave using the experimental dipolar couplings and resonance assignments in Fig. 3B; (E) structure calculated from the experimental data and resonance assignments in Fig. 3B; (F) superposition of 10 structural fits to the data in Fig. 3B with no assignment information; (G) superposition of 10 structural fits to the data in Fig. 3B with three residue type assignments; (H) ideal α-helix with a 12° tilt corresponding to the PISA wheel in Fig. 3C.

Fig. 4

Pentameric M2 ion channel in lipid bilayers (Opella et al. 1999)