Solid state NMR strategy for characterizing native membrane protein structures

Abstract

Unlike water soluble proteins, the structures of helical transmembrane proteins depend on a very complex environment. These proteins sit in the midst of dramatic electrical and chemical gradients and are often subject to variations in the lateral pressure profile, order parameters, dielectric constant, and other properties. Solid state NMR is a collection of tools that can characterize high resolution membrane protein structure in this environment. Indeed, prior work has shown that this complex environment significantly influences transmembrane protein structure. Therefore, it is important to characterize such structures under conditions that closely resemble its native environment. Researchers have used two approaches to gain protein structural restraints via solid state NMR spectroscopy. The more traditional approach uses magic angle sample spinning to generate isotropic chemical shifts, much like solution NMR. As with solution NMR, researchers can analyze the backbone chemical shifts to obtain torsional restraints. They can also examine nuclear spin interactions between nearby atoms to obtain distances between atomic sites. Unfortunately, for membrane proteins in lipid preparations, the spectral resolution is not adequate to obtain complete resonance assignments. Researchers have developed another approach for gaining structural restraints from membrane proteins: the use of uniformly oriented lipid bilayers, which provides a method for obtaining high resolution orientational restraints. When the bilayers are aligned with respect to the magnetic field of the NMR spectrometer, researchers can obtain orientational restraints in which atomic sites in the protein are restrained relative to the alignment axis. However, this approach does not allow researchers to determine the relative packing between helices. By combining the two approaches, we can take advantage of the information acquired from each technique to minimize the challenges and maximize the quality of the structural results. By combining the distance, torsional, and orientational restraints, we can characterize high resolution membrane protein structure in native-like lipid bilayer environments.

Figure 1

From an ¹⁵N labeled site, such as in the inset image of a peptide plane from a TM helix, the component of the chemical shift tensor (δ_ii) and dipolar interaction (ν_NH) parallel to the magnetic field (B_o) can be assessed as structural restraints.

Figure 2

Initial analysis of PISEMA spectra. A) PISEMA spectrum of CrgA ¹⁵N Phe labeled sample (residues: 33, 37, 51, 79 & 81). B) Calculated PISA wheels for different helical tilt angles. T=15° is consistent with the CrgA data in A and 38° is consistent with the data in C. C) PISEMA spectrum of Rv1861 ¹⁵N Val labeled protein.

Figure 3

The uniform oscillation of the anisotropic chemical shift (C) and dipolar interactions (A,B & D) is displayed more clearly with these wave patterns with exactly 3.6 residues per cycle. CrgA has two helices (A&B). Rv1861 has three helices, but only the data from helix #1 is displayed here (C&D).

Figure 4

Experimental ρ values from PISA wheel analysis are plotted against predicted values (100°/residue) for the same helix #1 residues of Rv1861 shown in Fig. 3 C&D.

Figure 5

DARR (Dipolar Assisted Rotational Resonance) MAS ssNMR spectra (243K, 50ms mixing & 10kHz spinning) from three proteins and their mixing times: A) M2 protein, B) CrgA, and C) Rv1861. The aliphatic resonance envelopes for Leu (red), Val (green), Ile (blue) and Ala (cyan) are highlighted with dotted lines showing nearly identical positions for their resonance envelopes in the three spectra.

Figure 6

DARR MAS ssNMR spectra from M2 protein (residues 22–62) showing a number of crosspeaks correlated with interhelical distance restraints (arrows). A&B) 50 ms mixing time; C&D) 100 ms mixing time; E&F) 200 ms mixing time.

Figure 7

Images of the M2 (22–62) protein structure showing the sparse interhelical distance restraints that uniquely constrain the quaternary structure.