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Water wires are critical for the functioning of many membrane proteins, as in channels that conduct water, protons, and other ions. Here, in liquid crystalline lipid bilayers under symmetric environmental conditions, the selective hydrogen bonding interactions between eight waters comprising a water wire and a subset of 26 carbonyl oxygens lining the antiparallel dimeric gramicidin A channel are characterized by 17 O NMR spectroscopy at 35.2 T (or 1,500 MHz for 1 H) and computational studies. While backbone 15 N spectra clearly indicate structural symmetry between the two subunits, single site 17 O labels of the pore-lining carbonyls report two resonances, implying a break in dimer symmetry caused by the selective interactions with the water wire. The 17 O shifts document selective water hydrogen bonding with carbonyl oxygens that are stable on the millisecond timescale. Such interactions are supported by density functional theory calculations on snapshots taken from molecular dynamics simulations. Water hydrogen bonding in the pore is restricted to just three simultaneous interactions, unlike bulk water environs. The stability of the water wire orientation and its electric dipole leads to opposite charge-dipole interactions for K + ions bound at the two ends of the pore, thereby providing a simple explanation for an ∼20-fold difference in K + affinity between two binding sites that are ∼24 Å apart. The 17 O NMR spectroscopy reported here represents a breakthrough in high field NMR technology that will have applications throughout molecular biophysics, because of the acute sensitivity of the 17 O nucleus to its chemical environment., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The National High Magnetic Field Laboratory has brought to field a Series-Connected Hybrid magnet for NMR spectroscopy. As a DC powered magnet it can be operated at fields up to 36.1T. The series connection between a superconducting outsert and a resistive insert dramatically minimizes the high frequency fluctuations of the magnetic field typically observed in purely resistive magnets. Current-density-grading among various resistive coils was used for improved field homogeneity. The 48mm magnet bore and 42mm outer diameter of the probes leaves limited space for conventional shims and consequently a combination of resistive and ferromagnetic shims are used. Field maps corrected for field instabilities were obtained and shimming achieved better than 1ppm homogeneity over a cylindrical volume of 1cm diameter and height. The magnetic field is regulated within 0.2ppm using an external 7 Li lock sample doped with paramagnetic MnCl 2 . The improved field homogeneity and field regulation using a modified AVANCE NEO console enables NMR spectroscopy at 1 H frequencies of 1.0, 1.2 and 1.5GHz. NMR at 1.5GHz reflects a 50% increase in field strength above the highest superconducting magnets currently available. Three NMR probes have been constructed each equipped with an external lock rf coil for field regulation. Initial NMR results obtained from the SCH magnet using these probes illustrate the very exciting potential of ultra-high magnetic fields., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high-resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water-soluble proteins and other membrane proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

Polarization Inversion Spin Exchange at Magic Angle (PISEMA) is a powerful experiment for determining peptide orientation in uniformly aligned samples such as planar membranes. In this paper, we present (14)N-PISEMA experiment which correlates (14)N quadrupolar coupling and (14)N-(1)H dipolar coupling. (14)N-PISEMA enables the use of (14)N quadrupolar coupling tensor as an ultra sensitive probe for peptide orientation and can be carried out without the need of isotope enrichment. The experiment is based on selective spin-exchange between a proton and a single-quantum transition of (14)N spins. The spin-exchange dynamics is described and the experiment is demonstrated with a natural abundant N-acetyl valine crystal sample.

We report here the backbone assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis. The backbone resonance assignments were determined based on triple-resonance experiments with uniformly [13C,15N]-labeled protein in LMPG detergent micelles.

The interactions of (15)N-labeled amantadine, an antiinfluenza A drug, with DMPC bilayers were investigated by solid-state NMR and by a 12.6-ns molecular dynamics (MD) simulation. The drug was found to assume a single preferred orientation and location when incorporated in these bilayers. The experimental and MD computational results demonstrate that the long axis of amantadine is on average parallel to the bilayer normal, and the amine group is oriented toward the headgroups of the lipid bilayers. The localization of amantadine was determined by paramagnetic relaxation and by the MD simulation showing that amantadine is within the interfacial region and that the amine interacts with the lipid headgroup and glycerol backbone, while the hydrocarbon portion of amantadine interacts with the glycerol backbone and much of the fatty acyl chain as it wraps underneath the drug. The lipid headgroup orientation changes on drug binding as characterized by the anisotropy of (31)P chemical shielding and (14)N quadrupolar interactions and by the MD simulation.

Membrane protein function within the membrane interstices is achieved by mechanisms that are not typically available to water-soluble proteins. The whole balance of molecular interactions that stabilize three-dimensional structure in the membrane environment is different from that in an aqueous environment. As a result interhelical interactions are often dominated by non-specific van der Waals interactions permitting dynamics and conformational heterogeneity in these interfaces. Here, solid-state NMR data of the transmembrane domain of the M2 protein from influenza A virus are used to exemplify such conformational plasticity in a tetrameric helical bundle. Such data lead to very high resolution structural restraints that can identify both subtle and substantial structural differences associated with various states of the protein. Spectra from samples using two different preparation protocols, samples prepared in the presence and absence of amantadine, and spectra as a function of pH are used to illustrate conformational plasticity.

A new two-dimensional scheme is proposed for accurate measurements of high-resolution chemical shifts and heteronuclear dipolar couplings in NMR of aligned samples. Both the (1)H chemical shifts and the (1)H-(15)N dipolar couplings are evolved in the indirect dimension while the (15)N chemical shifts are detected. This heteronuclear correlation (HETCOR) spectroscopy yields high-resolution (1)H chemical shifts split by the (1)H-(15)N dipolar couplings in the indirect dimension and the (15)N chemical shifts in the observed dimension. The advantages of the HETCOR technique are illustrated for a static (15)N-acetyl-valine crystal sample and a (15)N-labeled helical peptide sample aligned in hydrated lipid bilayers.

Li(+) and Ca(2+) binding to the carbonyl oxygen sites of a model peptide system has been studied by (17)O solid-state NMR spectroscopy. (17)O chemical shift (CS) and quadrupole coupling (QC) tensors are determined in four Gly-(Gly-(17)O)-Gly polymorphs by a combination of stationary and fast magic-angle spinning (MAS) methods at high magnetic field, 19.6 T. In the crystal lattice, the carbonyl oxygen of the central glycyl residue in two gly-gly-gly polymorphs form intermolecular hydrogen bonds with amides, whereas the corresponding carbonyl oxygens of the other two polymorphs form interactions with Li(+) and Ca(2+) ions. This permits a comparison of perturbations on (17)O NMR properties by ion binding and intermolecular hydrogen bonding. High quality spectra are augmented by density functional theory (DFT) calculations on large molecular clusters to gain additional theoretical insights and to aid in the spectral simulations. Ion binding significantly decreases the two (17)O chemical shift tensor components in the peptide plane, delta(11) and delta(22), and, thus, a substantial change in the isotropic chemical shift. In addition, quadrupole coupling constants are decreased by up to 1 MHz. The effects of ion binding are found to be almost an order of magnitude greater than those induced by hydrogen bonding.

The field of structural biology is becoming increasingly important as new technological developments facilitate the collection of data on the atomic structures of proteins and nucleic acids. The solid-state NMR method is a relatively new biophysical technique that holds particular promise for determining the structures of peptides and proteins that are located within the cell membrane. This method provides information on the orientation of the peptide planes relative to an external magnetic field. In this article, we discuss some of the mathematical methods and tools that are useful in deriving the atomic structure from these orientational data. We first discuss how the data are viewed as tensors, and how these tensors can be used to construct an initial atomic model, assuming ideal stereochemistry. We then discuss methods for refining the models using global optimization, with stereochemistry constraints treated as penalty functions. These two processes, initial model building followed by refinement, are the two crucial steps between data collection and the final atomic model.

Continuous-wave, ramped amplitude, and frequency modulated cross-polarization schemes (abbreviated as CWCP, RACP, and FMCP, respectively) are evaluated for static samples in anisotropic phases, such as peptides oriented in lipid environments. It is shown experimentally that both RACP and FMCP give rise to 20% higher polarized signal intensity in comparison to CWCP. The CP matching bandwidths for CWCP and RACP are about the same. Because of its adiabaticity, FMCP has a much broader CP matching bandwidth than CWCP and RACP. In addition, the (15)N RF amplitude used at the center of the FMCP matching profile is much lower than that of the CWCP and RACP matching profiles. A sample of [(15)N]Leu(4) labeled gramicidin A oriented in lipid bilayers was used to demonstrate these experiments.

The response of membrane-associated peptides toward the lipid environment or other binding partners can be monitored by solid-state NMR of suitably labeled side chains. Tryptophan is a prominent amino acid in transmembrane helices, and its (19)F-labeled analogues are generally biocompatible and cause little structural perturbation. Hence, we use 5F-Trp as a highly sensitive NMR probe to monitor the conformation and dynamics of the indole ring. To establish this (19)F-NMR strategy, gramicidin A was labeled with 5F-Trp in position 13 or 15, whose chi(1)/chi(2) torsion angles are known from previous (2)H-NMR studies. First, the alignment of the (19)F chemical shift anisotropy tensor within the membrane was deduced by lineshape analysis of oriented samples. Next, the three principal axes of the (19)F chemical shift anisotropy tensor were assigned within the molecular frame of the indole ring. Finally, determination of chi(1)/chi(2) for 5F-Trp in the lipid gel phase showed that the side chain alignment differs by up to 20 degrees from its known conformation in the liquid crystalline state. The sensitivity gain of (19)F-NMR and the reduction in the amount of material was at least 10-fold compared with previous (2)H-NMR studies on the same system and 100-fold compared with (15)N-NMR.

The effect of the Hartmann-Hahn mismatch delta = omega(eff)-omega(1S) during polarization inversion spin exchange at the magic angle (PISEMA) has been investigated, where omega(eff) and omega(1S) represent the amplitudes of the 1H effective spin-locking field at the magic angle and the 15N RF spin-locking field, respectively. During the PISEMA evolution period, the exact Hartmann-Hahn match condition (i.e., delta = 0) yields a maximum dipolar scaling factor of 0.816 for PISEMA experiments, while any mismatch results in two different effective fields for the first and second half of each frequency switched Lee-Goldburg (FSLG) cycle. The mismatch effect on the scaling factor depends strongly on the transition angle from one effective field to the other within each FSLG cycle as well as on the cycle time. At low RF spin-lock amplitudes in which the FSLG cycle time is relatively long, the scaling factor rapidly becomes smaller as omega(1S) becomes greater than omega(eff). On the other hand, when omega(1S) < omega(eff), there is relatively little effect on the scaling factor with variation in delta. As a result, the presence of RF inhomogeneities may significantly broaden the line-width in the dipolar dimension because of the mismatch effect. Higher RF spin-lock amplitudes result in a relatively small variation for the scaling factor. Furthermore, ramped amplitude of the 15N RF spin-lock field in synchronization with the flip-flop of the FSLG sequence minimizes the transition angle between the two effective fields within the FSLG cycle. It is shown experimentally that such a ramped amplitude not only gives rise to the same scaling factor but also results in a narrower dipolar line-width in comparison with the rectangular amplitude., (Copyright 2002 Elsevier Science (USA))

The spin dynamics of NMR spin locking of proton magnetization under a frequency-switched Lee-Goldburg (FSLG) pulse sequence is investigated for a better understanding of the line-narrowing mechanism in PISEMA experiments. For the sample of oriented 15N(1,3,5,7)-labeled gramicidin A in hydrated DMPC bilayers, it is found that the spin-lattice relaxation time T(1rho)(H) in the tilted rotating frame is about five times shorter when the 1H magnetization is spin locked at the magic angle by the FSLG sequence compared to the simple Lee-Goldburg sequence. It is believed that the rapid phase alternation of the effective fields during the FSLG cycles results in averaging of the spin lock field so that the spin lock becomes less efficient. A FSLG supercycle has been suggested here to slow the phase alternation. It has been demonstrated experimentally that a modified PISEMA pulse sequence with such supercycles gives rise to about 30% line narrowing in the dipolar dimension in the PISEMA spectra compared to a standard PISEMA pulse sequence.