15N Polarization Research Articles

Altus using P 450 for making drug metabolites and optimizing drug leads

We investigate solid-state dynamic nuclear polarization of 13C and 15N nuclei using monoradical trityl OX063 as a polarizing agent in a magnetic field of 14.1 T with magic angle spinning at ∼100 K. We monitored the field dependence of direct 13C and 15N polarization for frozen [13C, 15N] urea and achieved maximum absolute enhancement factors of 240 and 470, respectively. The field profiles are consistent with polarization of 15N spins via either the solid effect or the cross effect, and polarization of 13C spins via a combination of cross effect and solid effect. For microcrystalline, 15N-enriched tryptophan synthase sample containing trityl radical, a 1500-fold increase in 15N signal was observed under microwave irradiation. These results show the promise of trityl radicals and their derivatives for direct polarization of low gamma, spin-½ nuclei at high magnetic fields and suggest a novel approach for selectively polarizing specific moieties or for polarizing systems which have low levels of protonation.

A Simple Approach to Membrane Protein Secondary Structure and Topology based on NMR Spectroscopy

This paper describes a simple, qualitative approach for the determination of membrane protein secondary structure and topology in lipid bilayer membranes. The approach is based on the observation of wheel-like resonance patterns observed in the NMR 1H- 15N/ 15N polarization inversion with spin exchange at the magic angle (PISEMA) and 1H/ 15N heteronuclear correlation (HETCOR) spectra of membrane proteins in oriented lipid bilayers. These patterns, named Pisa wheels, have been previously shown to reflect helical wheel projections of residues that are characteristic of α-helices associated with membranes. This study extends the analysis of these patterns to β-strands associated with membranes and demonstrates that, as for the case of α-helices, Pisa wheels are extremely sensitive to the tilt, rotation, and twist of β-strands in the membrane. Therefore, the Pisa wheels provide a sensitive, visually accessible, qualitative index of membrane protein secondary structure and topology.

Transmembrane Domain of M2 Protein from Influenza A Virus Studied by Solid-State 15N Polarization Inversion Spin Exchange at Magic Angle NMR

The M2 protein from the influenza A virus forms a proton channel in the virion that is essential for infection. This tetrameric protein appears to form a four-helix bundle spanning the viral membrane. Here the solid-state NMR method, 2D polarization inversion spin exchange at magic angle (PISEMA), has been used to obtain multiple constraints from specifically amino acid-labeled samples. The improvement of spectral resolution from 2D PISEMA over 1D methods and 2D separated local field methods is substantial. The reliability of the method is validated by comparison of anisotropic chemical shift and heteronuclear dipolar interactions from single site labeled samples. The quantitative interpretation of the high-resolution constraints confirms the helix tilt to be within the range of previous experimental determinations (32°–38°). The binding of the channel inhibitor, amantadine, results in no change in the backbone structure at position Val 27,28, which is thought to be a potential binding site for the inhibitor.

Heteronuclear cross polarization in liquid-state nuclear magnetic resonance: Mismatch compensation and relaxation behavior

Nuclear spin dynamics in liquid-state heteronuclear cross-polarization experiments were explored by experiments on di-15N -labeled histidine in DMSO-d6. Compensation for Hartmann–Hahn mismatch was achieved by simultaneously shifting the phases of both spin-locking fields by π at intervals short compared to the spin–spin coupling. With rf compensation, slightly more 15N polarization was achieved than with the conventional selective INEPT scheme. This was attributed to relaxation by slowly fluctuating random fields associated with proton exchange. Theory for the rf mismatch compensation and relaxation behavior of the Hartmann–Hahn method is presented for the case of a heteronuclear 2-spin system.