H/C/N triple-resonance experiments for structure determinaton of membrane proteins by oriented-sample NMR.

The benefits of triple-resonance experiments for structure determination of macroscopically oriented membrane proteins by solid-state NMR are discussed. While double-resonance H/N experiments are effective for structure elucidation of alpha-helical domains, extension of the method of oriented samples to more complex topologies and assessing side-chain conformations necessitates further development of triple-resonance (H/C/N) NMR pulse sequences. Incorporating additional spectroscopic dimensions involving C spin-bearing nuclei, however, introduces essential complications arising from the wide frequency range of the H-C dipolar couplings and C CSA (>20 ​kHz), and the presence of the C-C homonuclear dipole-dipole interactions.

NMR "Crystallography" for Uniformly ( C, N)-Labeled Oriented Membrane Proteins.

In oriented-sample (OS) solid-state NMR of membrane proteins, the angular-dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only H- N dipolar couplings and N chemical shifts have been routinely assessed in oriented N-labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints.

Protein Rotational Dynamics in Aligned Lipid Membranes Probed by Anisotropic T NMR Relaxation.

A membrane-bound form of Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles was used as a molecular probe to quantify for the viscosity of the lipid membrane interior by measuring the uniaxial rotational diffusion coefficient of the protein. Orientationally dependent N NMR relaxation times in the rotating frame, or T, were determined by fitting individually the decay of the resolved NMR peaks corresponding to the transmembrane helix of Pf1 coat protein as a function of the spin-lock time incorporated into the 2D SAMPI4 pulse sequence. The T relaxation mechanism was modeled by uniaxial rotational diffusion on a cone, which yields a linear correlation with respect to the bond factor sinθ, where θ is the angle that the NH bond forms with respect to the axis of rotation.

Sensitivity enhancement for membrane proteins reconstituted in parallel and perpendicular oriented bicelles obtained by using repetitive cross-polarization and membrane-incorporated free radicals.

Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.