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The availability of 3D printers and an assortment of polymers that can be fashioned into a wide variety of shapes provides opportunities to rethink the design and construction of probes for NMR spectroscopy. The direct interfacing of computer aided design (CAD) with precise 3D printing enables the simplification and optimization of probes through the rapid production of components. Here we demonstrate the use of 3D printing to fully integrate a permanent former for the radiofrequency (RF) coil with the sample chamber (equivalent to the sample tube). This simultaneously increases the sample volume and improves the filling factor within a fixed outer diameter (OD). It also reduces the space lost in dual coil arrangements where a high frequency resonator is positioned outside a solenoid coil tuned to one or more lower frequencies, making multiple-resonance experiments more efficient. The initial applications demonstrate the possibilities for future designs that reimagine the interface between resonators and the liquid, solid, and heterogeneous samples encountered in NMR studies of biomolecules, polymers, surfaces, and spectroscopy (MRS) and imaging (MRI) of biological organs and intact organisms.

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

Well-hydrated phospholipid bilayers provide a near-native environment for membrane proteins. They enable the preparation of chemically-defined samples suitable for NMR and other spectroscopic experiments that reveal the structure, dynamics, and functional interactions of the proteins at atomic resolution. The synthetic polymer styrene maleic acid (SMA) can be used to prepare detergent-free samples that form macrodiscs with diameters greater than 30 nm at room temperature, and spontaneously align in the magnetic field of an NMR spectrometer at temperatures above 35 °C. Here we show that magnetically aligned macrodiscs are particularly well suited for solid-state NMR experiments of membrane proteins because the SMA-lipid assembly both immobilizes the embedded protein and provides uniaxial order for oriented sample (OS) solid-state NMR studies. We show that aligned macrodiscs incorporating four different membrane proteins with a wide range of sizes and topological complexity yield high-resolution OS solid-state NMR spectra. The work is dedicated to Michelle Auger who made key contributions to the field of membrane and membrane protein biophysics.

Oriented sample solid-state NMR is a complementary approach to protein structure determination with the distinct advantage that it can be applied to supramolecular assemblies, such as viruses and membrane proteins, under near-native conditions, which generally include high levels of hydration as found in living systems. Thus, in order to perform 1H detected versions of multi-dimensional experiments water suppression techniques must be integrated into the pulse sequences. For example, 1H-windowed detection of 1H-15N dipolar couplings enable multi-dimensional NMR experiments to be performed. Here we show that the addition of a solvent suppression pulse during the z-filter interval greatly improves the sensitivity of the experiments by suppressing the 1H signals from water present. This is demonstrated here with a crystal sample submerged in water and then extended to proteins. The combination of solvent-suppressed 1H detected PISEMO and the use of a strip shield-solenoid coil probe configuration provides a two-fold sensitivity enhancement in both the crystal sample and Pf1 coat protein sample compared to the 15N direct detection method. Here we also examine protein NMR line-widths and sensitivity enhancements in the context of window detected separated local field experiments for protein samples.

Extensive deuteration can be used to simplify NMR spectra by "diluting" and minimizing the effects of the abundant 1H nuclei. In solution-state NMR and magic angle spinning solid-state NMR of proteins, perdeuteration has been widely applied and its effects are well understood. Oriented sample solid-state NMR of proteins, however, is at a much earlier stage of development. In spite of the promise of the approach, the effects of sample deuteration are largely unknown. Here we map out the effects of perdeuteration on solid-state NMR spectra of aligned samples by closely examining differences in results obtained on fully protiated and perdeuterated samples, where all of the carbon sites have either 1H or 2H bonded to them, respectively. The 2H and 15N labeled samples are back-exchanged in 1H2O solution so that the amide 15N sites have a bonded 1H. Line-widths in the 15N chemical shift, 1H chemical shift, and 1H-15N dipolar coupling frequency dimensions were compared for peptide single crystals as well as membrane proteins aligned along with the phospholipids in bilayers with their normals perpendicular to the direction of the magnetic field. Remarkably, line-width differences were not found between fully protiated and perdeuterated samples. However, in the absence of effective 1H-1H homonuclear decoupling, the line-widths in the 1H-15N heteronuclear dipolar coupling frequency dimension were greatly narrowed in the perdeuterated samples. In proton-driven spin diffusion (PDSD) experiments, no effects of perdeuteration were observed. In contrast, in mismatched Hartmann-Hahn experiments, perdeuteration enhances cross-peak intensities by allowing more efficient spin-exchange with less polarization transfer back to the carbon-bound 1H. Here we show that in oriented sample solid-state NMR, the effects of perdeuteration can be exploited in experiments where 1H-1H homonuclear decoupling cannot be applied. These data also provide evidence for the possible contribution of direct 15N-15N dilute-spin mixing mechanism in proton-driven spin diffusion experiments.

Macrodiscs, which are magnetically alignable lipid bilayer discs with diameters of >30 nm, were obtained by solubilizing protein-containing liposomes with styrene-maleic acid copolymers. Macrodiscs provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under physiological conditions. The narrow resonance linewidths observed from membrane proteins in styrene-maleic acid macrodiscs advance structure determination by oriented sample solid-state NMR spectroscopy.

The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G protein-coupled receptor, located in the cell membrane of neutrophils. Here, we describe 1H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn2+ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

The structure of monomeric human chemokine IL-8 (residues 1-66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1-72), with the main differences being the location of the N-loop (residues 10-22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67-72). NMR was used to analyze the interactions of monomeric IL-8 (1-66) with ND-CXCR1 (residues 1-38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1-72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

We describe high resolution MAS solid-state NMR experiments that utilize (1)H detection with 60kHz magic angle spinning; simultaneous cross-polarization from (1)H to (15)N and (13)C nuclei; bidirectional cross-polarization between (13)C and (15)N nuclei; detection of both amide nitrogen and aliphatic carbon (1)H; and measurement of both (13)C and (15)N chemical shifts through multi-dimensional correlation experiments. Three-dimensional experiments correlate amide (1)H and alpha (1)H selectively with (13)C or (15)N nuclei in a polypeptide chain. Two separate three-dimensional spectra correlating (1)Hα/(13)Cα/(1)H(N) and (1)H(N)/(15)N/(1)Hα are recorded simultaneously in a single experiment, demonstrating that a twofold savings in experimental time is potentially achievable. Spectral editing using bidirectional coherence transfer pathways enables simultaneous magnetization transfers between (15)N, (13)Cα(()(i)()) and (13)C'(()(i)(-1)), facilitating intra- and inter-residue correlations for sequential resonance assignment. Non-uniform sampling is integrated into the experiments, further reducing the length of experimental time.

Virus protein U (Vpu) from HIV-1, a small membrane protein composed of a transmembrane helical domain and two α-helices in an amphipathic cytoplasmic domain, down modulates several cellular proteins, including CD4, BST-2/CD317/tetherin, NTB-A, and CCR7. The interactions of Vpu with these proteins interfere with the immune system and enhance the release of newly synthesized virus particles. It is essential to characterize the structure and dynamics of Vpu in order to understand the mechanisms of the protein-protein interactions, and potentially to discover antiviral drugs. In this article, we describe investigations of the cytoplasmic domain of Vpu as well as full-length Vpu by NMR spectroscopy. These studies are complementary to earlier analysis of the transmembrane domain of Vpu. The results suggest that the two helices in the cytoplasmic domain form a U-shape. The length of the inter-helical loop in the cytoplasmic domain and the orientation of the third helix vary with the lipid composition, which demonstrate that the C-terminal helix is relatively flexible, providing accessibility for interaction partners.

Type II nonribosomal peptide synthetases (NRPS) generate exotic amino acid derivatives that, combined with additional pathways, form many bioactive natural products. One family of type II NRPSs produce pyrrole moieties, which commonly arise from proline oxidation while tethered to a conserved, type II peptidyl carrier protein (PCP), as exemplified by PltL in the biosynthesis of pyoluteorin. We sought to understand the structural role of pyrrole PCPs in substrate and protein interactions through the study of pyrrole analogs tethered to PltL. Solution-phase NMR structural analysis revealed key interactions in residues of helix II and III with a bound pyrrole moiety. Conservation of these residues among PCPs in other pyrrole containing pathways suggests a conserved mechanism for formation, modification, and incorporation of pyrrole moieties. Further NOE analysis provided a unique pyrrole binding motif, offering accurate substrate positioning within the cleft between helices II and III. The overall structure resembles other PCPs but contains a unique conformation for helix III. This provides evidence of sequestration by the PCP of aromatic pyrrole substrates, illustrating the importance of substrate protection and regulation in type II NRPS systems.

The highly anisotropic environment of the lipid bilayer membrane imposes significant constraints on the structures and functions of membrane proteins. However, NMR structure calculations typically use a simple repulsive potential that neglects the effects of solvation and electrostatics, because explicit atomic representation of the solvent and lipid molecules is computationally expensive and impractical for routine NMR-restrained calculations that start from completely extended polypeptide templates. Here, we describe the extension of a previously described implicit solvation potential, eefxPot, to include a membrane model for NMR-restrained calculations of membrane protein structures in XPLOR-NIH. The key components of eefxPot are an energy term for solvation free energy that works together with other nonbonded energy functions, a dedicated force field for conformational and nonbonded protein interaction parameters, and a membrane function that modulates the solvation free energy and dielectric screening as a function of the atomic distance from the membrane center, relative to the membrane thickness. Initial results obtained for membrane proteins with structures determined experimentally in lipid bilayer membranes show that eefxPot affords significant improvements in structural quality, accuracy, and precision. Calculations with eefxPot are straightforward to implement and can be used to both fold and refine structures, as well as to run unrestrained molecular-dynamics simulations. The potential is entirely compatible with the full range of experimental restraints measured by various techniques. Overall, it provides a useful and practical way to calculate membrane protein structures in a physically realistic environment.

Many viruses express small hydrophobic membrane proteins. These proteins are often referred to as viroporins because they exhibit ion channel activity. However, the channel activity has not been definitively associated with a biological function in all cases. More generally, protein-protein and protein-phospholipid interactions have been associated with specific biological activities of these proteins. As research has progressed there is a decreased emphasis on potential roles of the channel activity, and increased research on multiple other biological functions. This being the case, it may be more appropriate to refer to them as 'viral membrane-spanning miniproteins'. Structural studies are illustrated with Vpu from HIV-1 and p7 from HCV.

The restriction factor BST2 (tetherin) prevents the release of enveloped viruses from the host cell and is counteracted by HIV-1 Vpu. Vpu and BST2 interact directly via their transmembrane domains. This interaction enables Vpu to induce the surface down-regulation and the degradation of BST2, but neither of these activities fully accounts for the ability of Vpu to enhance virion release. During a study of naturally occurring Vpu proteins, we found that a tryptophan residue near the Vpu C terminus is particularly important for enhancing virion release. Vpu proteins with a W76G polymorphism degraded and down-regulated BST2 from the cell surface, yet they inefficiently stimulated virion release. Here we explore the mechanism of this anomaly. We find that Trp-76 is critical for the ability of Vpu to displace BST2 from sites of viral assembly in the plane of the plasma membrane. This effect does not appear to involve a general reorganization of the membrane microdomains associated with virion assembly, but rather is a specific effect of Vpu on BST2. Using NMR spectroscopy, we find that the cytoplasmic domain of Vpu and Trp-76 specifically interact with lipids. Moreover, paramagnetic relaxation enhancement studies show that Trp-76 inserts into the lipid. These data are consistent with a model whereby Trp-76 anchors the C terminus of the cytoplasmic tail of Vpu to the plasma membrane, enabling the movement of Vpu-bound BST2 away from viral assembly sites.

The native environment for a membrane protein is a phospholipid bilayer. Because the protein is immobilized on NMR timescales by the interactions within a bilayer membrane, solid-state NMR methods are essential to obtain high-resolution spectra. Approaches have been developed for both unoriented and oriented samples, however, they all rest on the foundation of the most fundamental aspects of solid-state NMR, and the chemical shift and homo- and hetero-nuclear dipole-dipole interactions. Solid-state NMR has advanced sufficiently to enable the structures of membrane proteins to be determined under near-native conditions in phospholipid bilayers.

The motional averaging of powder pattern line shapes is one of the most fundamental aspects of sold-state NMR. Since membrane proteins in liquid crystalline phospholipid bilayers undergo fast rotational diffusion, all of the signals reflect the angles of the principal axes of their dipole-dipole and chemical shift tensors with respect to the axis defined by the bilayer normal. The frequency span and sign of the axially symmetric powder patterns that result from motional averaging about a common axis provide sufficient structural restraints for the calculation of the three-dimensional structure of a membrane protein in a phospholipid bilayer environment. The method is referred to as rotationally aligned (RA) solid-state NMR and demonstrated with results on full-length, unmodified membrane proteins with one, two, and seven trans-membrane helices. RA solid-state NMR is complementary to other solid-state NMR methods, in particular oriented sample (OS) solid-state NMR of stationary, aligned samples. Structural distortions of membrane proteins from the truncations of terminal residues and other sequence modifications, and the use of detergent micelles instead of phospholipid bilayers have also been demonstrated. Thus, it is highly advantageous to determine the structures of unmodified membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. RA solid-state NMR provides a general method for obtaining accurate and precise structures of membrane proteins under near-native conditions.

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.

The efficiency and selectivity of SPECIFIC-CP, a widely used method for selective double cross-polarization in triple-resonance magic angle spinning solid-state NMR, is improved by performing the tangential-shaped (13)C irradiation at an offset frequency that meets the Lee-Goldburg condition (LG-SPECIFIC-CP). This is demonstrated on polycrystalline samples of uniformly (13)C, (15)N labeled N-acetyl-leucine and N-formyl-Met-Leu-Phe-OH (MLF) at 700MHz and 900MHz (1)H resonance frequencies, respectively. For the single (13)Cα of N-acetyl-leucine, relative to conventional broad band cross-polarization, the SPECIFIC-CP signal has 47% of the intensity. Notably, the LG-SPECIFIC-CP signal has 72% of the intensity, essentially the theoretical maximum. There were no other changes in the experimental parameters. The three (13)Cα signals in MLF show some variation in intensities, reflecting the relatively narrow bandwidth of a frequency-offset procedure, and pointing to future developments for this class of experiment.

MerF is a mercury transport membrane protein from the bacterial mercury detoxification system. By performing a solid-state INEPT experiment and measuring chemical shift anisotropy frequencies in aligned samples, we are able to improve on the accuracy and precision of the initial structure that we presented. MerF has four N-terminal and eleven C-terminal residues that are mobile and unstructured in phospholipid bilayers. The structure presented here has average pairwise RMSDs of 1.78 Å for heavy atoms and 0.92 Å for backbone atoms.

Multiple acquisition spectroscopy (MACSY) experiments that enable multiple free induction decays to be recorded during individual experiments are demonstrated. In particular, the experiments incorporate separated local field spectroscopy into homonuclear and heteronuclear correlation spectroscopy. The measured heteronuclear dipolar couplings are valuable in structure determination as well as in enhancing resolution by providing an additional frequency axis. In one example four different three-dimensional spectra are obtained in a single experiment, demonstrating that substantial potential saving in experimental time is available when multiple multi-dimensional spectra are required as part of solid-state NMR studies.

The benefits of protein structure refinement in water are well documented. However, performing structure refinement with explicit atomic representation of the solvent molecules is computationally expensive and impractical for NMR-restrained structure calculations that start from completely extended polypeptide templates. Here we describe a new implicit solvation potential, EEFx (Effective Energy Function for XPLOR-NIH), for NMR-restrained structure calculations of proteins in XPLOR-NIH. The key components of EEFx are an energy term for solvation energy that works together with other nonbonded energy functions, and a dedicated force field for conformational and nonbonded protein interaction parameters. The initial results obtained with EEFx show that significant improvements in structural quality can be obtained. EEFx is computationally efficient and can be used both to fold and refine structures. Overall, EEFx improves the quality of protein conformation and nonbonded atomic interactions. Moreover, such benefits are accompanied by enhanced structural precision and enhanced structural accuracy, reflected in improved agreement with the cross-validated dipolar coupling data. Finally, implementation of EEFx calculations is straightforward and computationally efficient. Overall, EEFx provides a useful method for the practical calculation of experimental protein structures in a physically realistic environment.

A method for making resonance assignments in magic angle spinning solid-state NMR spectra of membrane proteins that utilizes the range of heteronuclear dipolar coupling frequencies in combination with conventional chemical shift based assignment methods is demonstrated. The Dipolar Assisted Assignment Protocol (DAAP) takes advantage of the rotational alignment of the membrane proteins in liquid crystalline phospholipid bilayers. Improved resolution is obtained by combining the magnetically inequivalent heteronuclear dipolar frequencies with isotropic chemical shift frequencies. Spectra with both dipolar and chemical shift frequency axes assist with resonance assignments. DAAP can be readily extended to three- and four-dimensional experiments and to include both backbone and side chain sites in proteins.

In the stationary, aligned samples used in oriented sample (OS) solid-state NMR, (1)H-(1)H homonuclear dipolar couplings are not attenuated as they are in magic angle spinning solid-state NMR; consequently, they are available for participation in dipolar coupling-based spin-exchange processes. Here we describe analytically the pathways of (15)N-(15)N spin-exchange mediated by (1)H-(1)H homonuclear dipolar couplings. The mixed-order proton-relay mechanism can be differentiated from the third spin assisted recoupling mechanism by setting the (1)H to an off-resonance frequency so that it is at the "magic angle" during the spin-exchange interval in the experiment, since the "magic angle" irradiation nearly quenches the former but only slightly attenuates the latter. Experimental spectra from a single crystal of N-acetyl leucine confirm that this proton-relay mechanism plays the dominant role in (15)N-(15)N dilute-spin-exchange in OS solid-state NMR in crystalline samples. Remarkably, the "forbidden" spin-exchange condition under "magic angle" irradiation results in (15)N-(15)N cross-peaks intensities that are comparable to those observed with on-resonance irradiation in applications to proteins. The mechanism of the proton relay in dilute-spin-exchange is crucial for the design of polarization transfer experiments.

Covariance spectroscopy (COV), a statistical method that provides increased sensitivity, can be applied to two-dimensional high-resolution solid-state NMR experiments, such as homonuclear spin-exchange spectroscopy. We the alternative States sampling scheme to the experimental time by 50%. By combining COV with other processing methods for non-uniform sampling (NUS), many different three-dimensional experiments can be performed with substantial increases in overall sensitivity. As an example, we show a three-dimensional homonuclear spin-exchange/separated-local-field (SLF) spectrum that enables the assignment of resonances and the measurement of structural restraints from a single experiment performed in a limited amount of time.

Most biological functions are carried out in supramolecular assemblies. As a result of their slow reorientation in solution, these assemblies have been resistant to the widely employed solution NMR approaches. The development of solid-state NMR to first of all overcome the correlation time problem and then obtain informative high-resolution spectra of proteins in supramolecular assemblies, such as virus particles and membranes, is described here. High resolution solid-state NMR is deeply intertwined with the history of NMR, and the seminal paper was published in 1948. Although the general principles were understood by the end of the 1950s, it has taken more than fifty years for instrumentation and experimental methods to become equal to the technical problems presented by the biological assemblies of greatest interest. It is now possible to obtain atomic resolution structures of viral coat proteins in virus particles and membrane proteins in phospholipid bilayers by oriented sample solid-state NMR methods. The development of this aspect of the field of solid-state NMR is summarized in this review article.

Oriented sample 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 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.

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.

Two-dimensional (15)N chemical shift/(1)H chemical shift and three-dimensional (1)H-(15)N dipolar coupling/(15)N chemical shift/(1)H chemical shift MAS solid-state NMR correlation spectra of the filamentous bacteriophage Pf1 major coat protein show single-site resolution in noncrystalline, intact-phage preparations. The high sensitivity and resolution result from (1)H detection at 600MHz under 50kHz magic angle spinning using ∼0.5mg of perdeuterated and uniformly (15)N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear (1)H-(15)N dipolar coupling frequency dimension is shown to select among (15)N resonances, which will be useful in structural studies of larger proteins where the resonances exhibit a high degree of overlap in multidimensional chemical shift correlation spectra.

We describe the incorporation of non-uniform sampling (NUS) compressed sensing (CS) into oriented sample (OS) solid-state NMR for stationary aligned samples and magic angle spinning (MAS) Solid-state NMR for unoriented 'powder' samples. Both simulated and experimental results indicate that 25-33% of a full linearly sampled data set is required to reconstruct two- and three-dimensional solid-state NMR spectra with high fidelity. A modest increase in signal-to-noise ratio accompanies the reconstruction.

Hydrophobic membrane-spanning helices often are flanked by interfacial aromatic or charged residues. In this paper, we compare the consequences of single Trp → Tyr substitutions at each interface for the properties of a defined transmembrane helix in the absence of charged residues. The choice of molecular framework is critical for these single-residue experiments because the presence of "too many" aromatic residues (more than one at either membrane-water interface) introduces excess dynamic averaging of solid state NMR observables. To this end, we compare the outcomes when changing W(5) or W(19), or both of them, to tyrosine in the well-characterized transmembrane peptide acetyl-GGALW(5)(LA)6LW(19)LAGA-amide ("GWALP23"). By means of solid-state (2)H and (15)N NMR experiments, we find that Y(19)GW(5)ALP23 displays similar magnitudes of peptide helix tilt as Y(5)GW(19)ALP23 and responds similarly to changes in bilayer thickness, from DLPC to DMPC to DOPC. The presence of Y(19) changes the azimuthal rotation angle ρ (about the helix axis) to a similar extent as Y(5), but in the opposite direction. When tyrosines are substituted for both tryptophans to yield GY(5,19)ALP23, the helix tilt angle is again of comparable magnitude, and furthermore, the preferred azimuthal rotation angle ρ is relatively unchanged from that of GW(5,19)ALP23. The extent of dynamic averaging increases marginally when Tyr replaces Trp. Yet, importantly, all members of the peptide family having single Tyr or Trp residues near each interface exhibit only moderate and not highly extensive dynamic averaging. The results provide important benchmarks for evaluating conformational and dynamic control of membrane protein function.

Abstract The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein 'u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.

Structure determination by solid-state NMR of proteins is rapidly advancing as a result of recent developments of samples, experimental methods, and calculations. There are a number of different solid-state NMR approaches that utilize stationary samples, aligned samples, or magic angle spinning of unoriented "powder" samples, and depending on the sample and the experimental method they can emphasize the measurement of distances or angles, ideally both, as sources of structural constraints. Multidimensional correlation spectroscopy of low-gamma nuclei such as (15)N and (13)C is an important step for making resonance assignments and measurements of angular restraints in membrane proteins. However, the efficiency of coherence transfer predominantly depends upon the strength of the dipole-dipole interaction, and this can vary from site to site and between sample alignments, for example, during the mixing of (13)C and (15)N magnetization in stationary aligned and in magic angle spinning samples. Here, we demonstrate that the efficiency of polarization transfer can be improved by using adiabatic demagnetization and remagnetization techniques on stationary aligned samples, and proton assisted insensitive nuclei cross-polarization in magic angle sample spinning samples. The adiabatic cross-polarization technique provides an alternative mechanism for spin-diffusion experiments correlating (15)N/(15)N and (15)N/(13)C chemical shifts over large distances. Improved efficiency in cross-polarization with 40-100% sensitivity enhancements is observed in proteins and single crystals, respectively. We describe solid-state NMR experimental techniques that are optimal for membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. The techniques are illustrated with data from single crystals both of peptides and of membrane proteins in phospholipid bilayers.

One of the main applications of solid-state NMR is to study the structure and dynamics of biopolymers, such as membrane proteins, under physiological conditions where the polypeptides undergo global motions as they do in biological membranes. The effects of NMR radiofrequency irradiations on nuclear spins are strongly influenced by these motions. For example, we previously showed that the MSHOT-Pi4 pulse sequence yields spectra with resonance line widths about half of those observed using the conventional pulse sequence when applied to membrane proteins undergoing rapid uniaxial rotational diffusion in phospholipid bilayers. In contrast, the line widths were not changed in microcrystalline samples where the molecules did not undergo global motions. Here, we demonstrate experimentally and describe analytically how some Hamiltonian terms are susceptible to sample motions, and it is their removal through the critical π/2 Z-rotational symmetry that confers the "motion adapted" property to the MSHOT-Pi4 pulse sequence. This leads to the design of separated local field pulse sequence "Motion-adapted SAMPI4" and is generalized to an approach for the design of decoupling sequences whose performance is superior in the presence of molecular motions. It works by cancelling the spin interaction by explicitly averaging the reduced Wigner matrix to zero, rather than utilizing the 2π nutation to average spin interactions. This approach is applicable to both stationary and magic angle spinning solid-state NMR experiments.

Hepatitis C virus (HCV) protein p7 plays an important role in the assembly and release of mature virus particles. This small 63-residue membrane protein has been shown to induce channel activity, which may contribute to its functions. p7 is highly conserved throughout the entire range of HCV genotypes, which contributes to making p7 a potential target for antiviral drugs. The secondary structure of p7 from the J4 genotype and the tilt angles of the helices within bilayers have been previously characterized by nuclear magnetic resonance (NMR). Here we describe the three-dimensional structure of p7 in short chain phospholipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine) micelles, which provide a reasonably effective membrane-mimicking environment that is compatible with solution NMR experiments. Using a combination of chemical shifts, residual dipolar couplings, and PREs, we determined the structure of p7 using an implicit membrane potential combining both CS-Rosetta decoys and Xplor-NIH refinement. The final set of structures has a backbone root-mean-square deviation of 2.18 Å. Molecular dynamics simulations in NAMD indicate that several side chain interactions might be taking place and that these could affect the dynamics of the protein. In addition to probing the dynamics of p7, we evaluated several drug-protein and protein-protein interactions. Established channel-blocking compounds such as amantadine, hexamethylene amiloride, and long alkyl chain iminosugar derivatives inhibit the ion channel activity of p7. It has also been shown that the protein interacts with HCV nonstructural protein 2 at the endoplasmic reticulum and that this interaction may be important for the infectivity of the virus. Changes in the chemical shift frequencies of solution NMR spectra identify the residues taking part in these interactions.

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.

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.