Aligned peptoid-based macrodiscs for structural studies of membrane proteins by oriented-sample NMR.

Development of a robust, uniform, and magnetically orientable lipid mimetic will undoubtedly advance solid-state NMR of macroscopically aligned membrane proteins. Here, we report on a novel lipid membrane mimetic based on peptoid belts. The peptoids, composed of 15 residues, were synthesized by alternating N-(2-phenethyl)glycine with N-(2-carboxyethyl)glycine residues at a 2:1 molar ratio. The chemically synthesized peptoids possess a much lower degree of polydispersity versus styrene-maleic acid polymers, thus yielding uniform discs. Moreover, the peptoid oligomers are more flexible and do not require a specific folding, unlike lipoproteins, in order to wrap around the hydrophobic membrane core. The NMR spectra measured for the membrane-bound form of Pf1 coat protein incorporated in this new lipid mimetics demonstrate a higher order parameter and uniform linewidths compared with the conventional bicelles and peptide-based macrodiscs. Importantly, unlike bicelles, the peptoid-based macrodiscs are detergent free.

1H/13C/15N 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 1H/15N 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 (1H/13C/15N) NMR pulse sequences. Incorporating additional spectroscopic dimensions involving 13C spin-bearing nuclei, however, introduces essential complications arising from the wide frequency range of the 1H-13C dipolar couplings and 13C CSA (>20 â€‹kHz), and the presence of the 13C-13C homonuclear dipole-dipole interactions. The recently reported ROULETTE-CAHA pulse sequence, in combination with the selective z-filtering, can be used to evolve the structurally informative 1H-13C dipolar coupling arising from the aliphatic carbons while suppressing the signals from the carbonyl and methyl regions. Proton-mediated magnetization transfer under mismatched Hartman-Hahn conditions (MMHH) can be used to correlate 13C and 15N nuclei in such triple-resonance experiments for the subsequent 15N detection. The recently developed pulse sequences are illustrated for n-acetyl Leucine (NAL) single crystal and doubly labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. An interesting observation is that in the case of 15N-labeled NAL measured at 13C natural abundance, the triple (1H/13C/15N) MMHH scheme predominantly gives rise to long-range intermolecular magnetization transfers from 13C to 15N spins; whereas direct Hartmann-Hahn 13C/15N transfer is entirely intramolecular. The presented developments advance NMR of oriented samples for structure determination of membrane proteins and liquid crystals.

NMR "Crystallography" for Uniformly (13 C, 15 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 1 H-15 N dipolar couplings and 15 N chemical shifts have been routinely assessed in oriented 15 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. We have developed a new experimental triple-resonance NMR technique, which was applied to uniformly doubly (15 N, 13 C)-labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible 1 Hα -13 Cα dipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α-helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α-helical transmembrane structure for Pf1 protein.

Protein Rotational Dynamics in Aligned Lipid Membranes Probed by Anisotropic T1ρ 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 15N NMR relaxation times in the rotating frame, or T1ρ, 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 T1ρ relaxation mechanism was modeled by uniaxial rotational diffusion on a cone, which yields a linear correlation with respect to the bond factor sin4θB, where θB is the angle that the NH bond forms with respect to the axis of rotation. Importantly, the bond factors can be independently measured from the dipolar couplings in the separated local-field SAMPI4 spectra. From this dependence, the value of the diffusion coefficient D|| = 8.0 × 105 s-1 was inferred from linear regression of the experimental T1ρ data even without any spectroscopic assignment. Alternatively, a close value of D|| = 7.7 × 105 s-1 was obtained by fitting the T1ρ relaxation data for the assigned NMR peaks of the transmembrane domain of Pf1 to a wavelike pattern as a function of residue number. The method illustrates the use of single-helix transmembrane peptides as molecular probes to assess the dynamic parameters of biological membranes by NMR relaxation in oriented lipid bilayers.

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 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.