NMR studies of p7 protein from hepatitis C virus

The p7 protein of hepatitis C virus (HCV) plays an important role in the viral lifecycle. Like other members of the viroporin family of small membrane proteins, the amino acid sequence of p7 is largely conserved over the entire range of genotypes, and it forms ion channels that can be blocked by a number of established channel-blocking compounds. Its characteristics as a membrane protein make it difficult to study by most structural techniques, since it requires the presence of lipids to fold and function properly. Purified p7 can be incorporated into phospholipid bilayers and micelles. Initial solid-state nuclear magnetic resonance (NMR) studies of p7 in 14-O-PC/6-O-PC bicelles indicate that the protein contains helical segments that are tilted approximately 10° and 25° relative to the bilayer normal. A truncated construct corresponding to the second transmembrane domain of p7 is shown to have properties similar to those of the full-length protein, and was used to determine that the helix segment tilted at 10° is in the C-terminal portion of the protein. The addition of the channel blocker amantadine to the full-length protein resulted in selective chemical shift changes, demonstrating that NMR has a potential role in the development of drugs targeted to p7

Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR

Oriented Sample (OS) solid-state NMR spectroscopy can be used to determine the three-dimensional structures of membrane proteins in magnetically or mechanically aligned lipid bilayers. The bottleneck for applying this technique to larger and more challenging proteins is making resonance assignments, which is conventionally accomplished through the preparation of multiple selectively isotopically labeled samples and performing an analysis of residues in regular secondary structure based on Polarity Index Slant Angle (PISA) Wheels and Dipolar Waves. Here we report the complete resonance assignment of the full-length mercury transporter, MerF, an 81-residue protein, which is challenging because of overlapping PISA Wheel patterns from its two trans-membrane helices, by using a combination of solid-state NMR techniques that improve the spectral resolution and provide correlations between residues and resonances. These techniques include experiments that take advantage of the improved resolution of the MSHOT4-Pi4/Pi pulse sequence; the transfer of resonance assignments through frequency alignment of heteronuclear dipolar couplings, or through dipolar coupling correlated isotropic chemical shift analysis; (15)N/(15)N dilute spin exchange experiments; and the use of the proton-evolved local field (PELF) experiment with isotropic shift analysis to assign the irregular terminal and loop regions of the protein, which is the major “blind spot” of the PISA Wheel/Dipolar Wave method

Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples

Solid-state NMR spectroscopy has been used successfully for characterizing the structure and dynamics of membrane proteins as well as their interactions with other proteins in lipid bilayers. such an environment is often necessary for achieving native-like structures. sample preparation is the key to this success. Here we present a detailed description of a robust protocol that results in high-quality membrane protein samples for both magic-angle spinning and oriented-sample solid-state NMR. the procedure is demonstrated using two proteins: CrgA (two transmembrane helices) and rv1861 (three transmembrane helices), both from Mycobacterium tuberculosis. the success of this procedure relies on two points. First, for samples for both types of NMR experiment, the reconstitution of the protein from a detergent environment to an environment in which it is incorporated into liposomes results in ‘complete’ removal of detergent. second, for the oriented samples, proper dehydration followed by rehydration of the proteoliposomes is essential. By using this protocol, proteoliposome samples for magic-angle spinning NMR and uniformly aligned samples (orientational mosaicity of <1°) for oriented-sample NMR can be obtained within 10 d