Side-chain conformation of the M2 transmembrane peptide proton channel of influenza a virus from 19F solid-state NMR

The M2 transmembrane peptide (M2TMP) of the influenza A virus forms a tetrameric helical bundle that acts as a proton-selective channel important in the viral life cycle. The side-chain conformation of the peptide is largely unknown and is important for elucidating the proton-conducting mechanism and the channel stability. Using a 19F spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain 19F-19F distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. 19F CODEX data at a key residue of the proton channel, Trp41, confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of approximately 11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous 15N NMR experiments and the backbone channel diameter has a narrow allowed range, this 19F distance constrains the Trp41 side-chain conformation to t90 (chi1 approximately 180 degrees , chi2 approximately 90 degrees ). This Trp41 rotamer, combined with a previously measured 15N-13C distance between His37 and Trp411, suggests that the His37 rotamer is t-160. The implication of the proposed (His37, Trp41) rotamers to the gating mechanism of the M2 proton channel is discussed. Binding of the antiviral drug amantadine to the peptide does not affect the F-F distance at Trp41. Interhelical 19F-19F distances are also measured at residues 27 and 38, each mutated to 4-19F-Phe. For V27F-M2TMP, the 19F-19F distances suggest a mixture of dimers and tetramers, whereas the L38F-M2TMP data indicate two tetramers of different sizes, suggesting side chain conformational heterogeneity at this lipid-facing residue. This work shows that 19F spin diffusion NMR is a valuable tool for determining long-range intermolecular distances that shed light on the mechanism of action and conformational heterogeneity of membrane protein oligomers.     

Determination of peptide oligomerization in lipid bilayers using 19F spin diffusion NMR

Aggregation or oligomerization is important for the function of many membrane peptides such as ion channels and antimicrobial peptides. However, direct proof of aggregation and the determination of the number of molecules in the aggregate have been difficult due to the lack of suitable high-resolution methods for membrane peptides. We propose a 19F spin diffusion magic-angle-spinning NMR technique to determine the oligomeric state of peptides bound to the lipid bilayer. Magnetization transfer between chemically equivalent but orientationally different 19F spins on different molecules reduces the 19F magnetization in an exchange experiment. At long mixing times, the equilibrium 19F magnetization is 1/M, where M is the number of orientationally different molecules in the aggregate. The use of the 19F spin increases the homonuclear dipolar coupling and thus the distance reach. We demonstrate this technique on crystalline model compounds with known numbers of molecules in the asymmetric unit cell, and show that 19F spin diffusion is more efficient than that of 13C by a factor of approximately 500. Application to a beta-hairpin antimicrobial peptide, protegrin-1, shows that the peptide is almost completely dimerized in POPC bilayers at a concentration of 7.4 mol %. Decreasing the peptide concentration reduced the dimer fraction. Using a monomer-dimer equilibrium model, we estimate the DeltaG for dimer formation to be -10.2 +/- 2.3 kJ/mol. This is in good agreement with the previously measured free energy reduction for partitioning and aggregating beta-sheet peptides into phospholipid membranes. This 19F spin diffusion technique opens the possibility of determining the oligomeric structures of membrane peptides.     

Structure, topology, and dynamics of membrane peptides and proteins from solid-state NMR spectroscopy

The high-resolution structure of membrane proteins is notoriously difficult to determine due to the hydrophobic nature of the protein-membrane complexes. Solid-state NMR spectroscopy is a unique and powerful atomic-resolution probe of the structure and dynamics of these important biological molecules. A number of new solid-state NMR methods for determining the depth of insertion, orientation, oligomeric structure, and long-range (10-15 A) distances of membrane proteins are summarized. Membrane protein depths can now be determined using several complementary techniques with varying site-specificity, distance precision, and mobility requirement on the protein. Membrane protein orientation can now be determined with or without macroscopic alignment, the latter providing a novel alternative for orientation determination of intrinsically curvature-inducing proteins. The novel analyses of beta-sheet membrane protein orientation are described. The quaternary structure of membrane peptide assemblies can now be elucidated using a 19F spin diffusion technique that simultaneously yields the oligomeric number and intermolecular distances up to 15 A. Finally, long-range distances up to approximately 10 A can now be measured using 1H spins with an accuracy of better than 1 A. These methods are demonstrated on several beta-sheet membrane peptides with antimicrobial activities and on two alpha-helical ion-channel proteins. Finally, we show that the nearly ubiquitous dynamics of membrane proteins can be readily examined using 2D correlation experiments. An intimate appreciation of molecular motion in these systems not only leads to important insights into the specific function of these membrane proteins but also may be exploited for other purposes such as orientation determination.     

Self-assembly of flexible β-strands into immobile amyloid-like β-sheets in membranes as revealed by solid-state 19F NMR

The cationic peptide [KIGAKI](3) was designed as an amphiphilic β-strand and serves as a model for β-sheet aggregation in membranes. Here, we have characterized its molecular conformation, membrane alignment, and dynamic behavior using solid-state (19)F NMR. A detailed structure analysis of selectively (19)F-labeled peptides was carried out in oriented DMPC bilayers. It showed a concentration-dependent transition from monomeric β-strands to oligomeric β-sheets. In both states, the rigid (19)F-labeled side chains project straight into the lipid bilayer but they experience very different mobilities. At low peptide-to-lipid ratios ≤1:400, monomeric [KIGAKI](3) swims around freely on the membrane surface and undergoes considerable motional averaging, with essentially uncoupled φ/ψ torsion angles. The flexibility of the peptide backbone in this 2D plane is reminiscent of intrinsically unstructured proteins in 3D. At high concentrations, [KIGAKI](3) self-assembles into immobilized β-sheets, which are untwisted and lie flat on the membrane surface as amyloid-like fibrils. This is the first time the transition of monomeric β-strands into oligomeric β-sheets has been characterized by solid-state NMR in lipid bilayers. It promises to be a valuable approach for studying membrane-induced amyloid formation of many other, clinically relevant peptide systems.     

Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy

A major challenge for the structure determination of integral membrane proteins by solution NMR spectroscopy is the limited number of NOE restraints in these systems stemming from extensive deuteration. Paramagnetic relaxation enhancement (PRE) by means of nitroxide spin-labels can provide valuable long-range distance information but, in practice, has limits in its application to membrane proteins because spin-labels are often incompletely reduced in highly apolar environments. Using the integral membrane protein OmpA as a model system, we introduce a method of parallel spin-labeling with paramagnetic and diamagnetic labels and show that distances in the range 15-24 Angstroms can be readily determined. The protein was labeled at 11 water-exposed and lipid-covered sites, and 320 PRE distance restraints were measured. The addition of these restraints resulted in significant improvement of the calculated backbone structure of OmpA. Structures of reasonable quality can even be calculated with PRE distance restraints only, i.e., in the absence of NOE distance restraints.     

Long-range 1H-19F distance measurement in peptides by solid-state NMR

A new NMR technique for determining long-range 1H-19F distances in solids is demonstrated. Using a modified rotational-echo double resonance (REDOR) sequence involving 1H homonuclear decoupling and composite 19F pulses, we show that it is possible to determine 1H-19F distances to approximately 8 A. The detrimental effect of the large 19F chemical shift to REDOR dephasing is partially compensated for by the composite pulse, 90 degrees 225 degrees 315 degrees . The 1HNLeu-19FPhe distance in the peptide f-MLF-OH was found to be 7.7 A. This was used to refine the Phe side chain conformation. The 1H-19F REDOR technique should be useful for restraining the three-dimensional structure of proteins.     

Centerband-Only Detection of Exchange NMR with Natural-Abundance Correction Reveals an Expanded Unit Cell in Phenylalanine Crystals

The NMR pulse sequence CODEX (centerband-only detection of exchange) is a widely used method to report on the number of magnetically inequivalent spins that exchange magnetization via spin diffusion. For crystals, this rules out certain symmetries, and the rate of equilibration is sensitive to distances. Here we show that for ¹³C CODEX, consideration of natural abundance spins is necessary for crystals of high complexity, demonstrated here with the amino acid phenylalanine. The NMR data rule out the C₂ space group that was originally reported for phenylalanine, and are only consistent with a larger unit cell containing eight magnetically inequivalent molecules. Such an expanded cell was recently described based on single crystal data. The large unit cell dictates the use of long spin diffusion times of more than 200 seconds, in order to equilibrate over the entire unit cell volume of 1622 ų.     

Improved pulse sequences for pure exchange solid-state NMR spectroscopy

Spin-exchange experiments are useful for improving the resolution and establishment of sequential assignments in solid-state NMR spectra of uniformly (15)N-labeled proteins oriented macroscopically in phospholipid bilayers. To exploit this advantage fully, it is crucial that the diagonal peaks in the two-dimensional exchange spectra are suppressed. This may be accomplished using the recent pure-exchange (PUREX) experiments, which, however, suffer from up to a threefold reduction of the cross-peak intensity relative to experiments without diagonal-peak suppression. This loss in sensitivity may severely hamper the applicability for the study of membrane proteins. In this paper, we present a two-dimensional exchange experiment (iPUREX) which improves the PUREX sensitivity by 50%. The performance of iPUREX is demonstrated experimentally by proton-mediated (15)N-(15)N spin-exchange experiments for a (15)N-labeled N-acetyl-L-valyl-L-leucine dipeptide. The relevance of exchange experiments with diagonal-peak suppression for large, uniformly (15)N-labeled membrane proteins in oriented phospholipid bilayers is demonstrated numerically for the G-protein coupled receptor rhodopsin.     

Measurement of Angstrom to Nanometer Molecular Distances with 19F Nuclear Spins by EPR/ENDOR Spectroscopy

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using ¹⁹F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the ¹⁹F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.     

NMR of membrane-associated peptides and proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.     

19F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins

Fluorine NMR paramagnetic relaxation enhancement was evaluated as a versatile approach for extracting distance information in selectively F‐labeled proteins. Proof of concept and initial applications are presented for the HIV‐inactivating lectin cyanovirin‐N. Single F atoms were introduced at the 4‐, 5‐, 6‐ or 7 positions of Trp49 and the 4‐position of Phe4, Phe54, and Phe80. The paramagnetic nitroxide spin label was attached to Cys residues that were placed into the protein at positions 50 or 52. ¹⁹F‐T₂ NMR spectra with different relaxation delays were recorded and the transverse ¹⁹F‐PRE rate, ¹⁹F‐Γ₂, was used to determine the average distance between the F nucleus and the paramagnetic center. Our data show that experimental ¹⁹F PRE‐based distances correspond to 0.93 of the ¹H_N‐PRE distances, in perfect agreement with the gyromagnetic γ¹⁹F/γ¹H ratio, thereby demonstrating that ¹⁹F PREs are excellent alternative parameters for quantitative distance measurements in selectively F‐labeled proteins.     

Mismatched Hartmann-Hahn conditions cause proton-mediated intermolecular magnetization transfer between dilute low-spin nuclei in NMR of static solids

Mismatched Hartmann-Hahn conditions between the protons and dilute spins (such as 15N) are found to cause intermolecular magnetization transfer between the low-gamma nuclei over long distances. This transfer is purely proton mediated and occurs even in the absence of direct 15N-15N couplings. This has been demonstrated experimentally using a static single crystal of n-acetyl Leucine with intermolecular distances between the 15N nuclei exceeding 6.5 A. A quantum-mechanical explanation of this phenomenon is given based on the average-Hamiltonian theory which was confirmed by detailed numerical many-spin simulations. The theory and experiment presented in the present paper may help in the development of solid-state NMR methods for studying interhelical contacts in membrane proteins, as well as for their spectral assignment.     

Site-specific incorporation of a (19)F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity

19F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biological interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, we have inserted trifluoromethyl-l-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19F NMR chemical shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Angstrom). 19F signal sensitivity and resolution was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.     

Modelling of repulsive potentials from atom charge density distributions: interactions of inert gas atoms

Using electron charge density overlap integrals, a correlation procedure for estimating short-range repulsive intermolecular potentials has been investigated. It is found that the repulsive potentials correlate well with the density overlap integral divided by the square of the distance. A simple combining rule for inert gas atoms is obtained and compared with an accurate determination of repulsive potentials for all the mixed rare gases and with other combining rules.     

Determining the orientation of uniaxially rotating membrane proteins using unoriented samples: a 2H, 13C, AND 15N solid-state NMR investigation of the dynamics and orientation of a transmembrane helical bundle

Membrane protein orientation has traditionally been determined by NMR using mechanically or magnetically aligned samples. Here we show a new NMR approach that abolishes the need for preparing macroscopically aligned membranes. When the protein undergoes fast uniaxial rotation around the bilayer normal, the 0 degrees -frequency of the motionally averaged powder spectrum is identical to the frequency of the aligned protein whose alignment axis is along the magnetic field. Thus, one can use unoriented membranes to determine the orientation of the protein relative to the bilayer normal. We demonstrate this approach on the M2 transmembrane peptide (M2TMP) of influenza A virus, which is known to assemble into a proton-conducting tetrameric helical bundle. The fast uniaxial rotational diffusion of the M2TMP helical bundle around the membrane normal is characterized via 2H quadrupolar couplings, C-H and N-H dipolar couplings, 13C chemical shift anisotropies, and 1H T1rho relaxation times. We then show that 15N chemical shift anisotropy and N-H dipolar coupling measured on these powder samples can be analyzed to yield precise tilt angles and rotation angles of the helices. The data show that the tilt angle of the M2TMP helices depends on the membrane thickness to reduce the hydrophobic mismatch. Moreover, the orientation of a longer M2 peptide containing both the transmembrane domain and cytoplasmic residues is similar to the orientation of the transmembrane domain alone, suggesting that the transmembrane domain regulates the orientation of this protein and that structural information obtained from M2TMP may be extrapolated to the longer peptide. This powder-NMR approach for orientation determination is generally applicable and can be extended to larger membrane proteins.     

Long-range 19F-15N distance measurements in highly-13C, 15N-enriched solid proteins with 19F-dephased REDOR shift (FRESH) spectroscopy

We present a novel rotational-echo double resonance (REDOR) method for detection of multiple (19)F-(15)N distances in solid proteins. The method is applicable to protein samples containing a single (19)F label, in addition to high levels of (13)C and (15)N enrichment. REDOR dephasing pulses are applied on the (19)F channel during an indirect constant time chemical shift evolution period on (15)N, and polarization is then transferred to (13)C for detection, with high-power (1)H decoupling throughout the sequence. This four-channel experiment reports site-specifically on (19)F-(15)N distances, with highly accurate determinations of approximately 5 A distances and detection of correlations arising from internuclear distances of at least 8 A. We demonstrate the method on the well-characterized 56-residue model protein GB1, where the sole tryptophan residue (Trp-43) has been labeled with 5-(19)F-Trp, in a bacterial growth medium also including (13)C-glucose and (15)N ammonium chloride. In GB1, 11 distances are determined, all agreeing within 20% of the X-ray structure distances. We envision the experiment will be utilized to measure quantitative long-range distances for protein structure determination.     

Direct detection of membrane channels from gels using water-in-oil droplet bilayers

We form planar lipid bilayers between an aqueous droplet and a hydrogel support immersed in a lipid-oil solution. By scanning the bilayer over the surface of an SDS-PAGE gel, we are able to directly detect membrane proteins from gels using single-channel recording. Using this technique, we are able to examine low levels of endogenous protein from cell extracts without the need for over-expression. We also use droplet bilayers to detect small molecules from hydrogels. The bilayers show enhanced stability compared to conventional planar lipid bilayers, and both bilayer size and position can be controlled during an experiment. Hydrogel scanning with droplet bilayers provides a new method for the discovery and characterization of ion channels with the potential for high-throughput screening.     

GdIII-19F Distance Measurements for Proteins in Cells by Electron-Nuclear Double Resonance

Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron-electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that Gd^III-¹⁹F Mims electron-nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in-cell ENDOR measurements, complemented with room temperature solution and in-cell Gd^III-¹⁹F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin-labeled with rigid Gd^III tags. The proteins were delivered into human cells via electroporation. The solution and in-cell derived Gd^III-¹⁹F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the Gd^III and ¹⁹F regions in the cell.     

Phosphate-mediated arginine insertion into lipid membranes and pore formation by a cationic membrane peptide from solid-state NMR

The insertion of charged amino acid residues into the hydrophobic part of lipid bilayers is energetically unfavorable yet found in many cationic membrane peptides and protein domains. To understand the mechanism of this translocation, we measured the (13)C-(31)P distances for an Arg-rich beta-hairpin antimicrobial peptide, PG-1, in the lipid membrane using solid-state NMR. Four residues, including two Arg's, scattered through the peptide were chosen for the distance measurements. Surprisingly, all residues show short distances to the lipid (31)P: 4.0-6.5 A in anionic POPE/POPG membranes and 6.5-8.0 A in zwitterionic POPC membranes. The shortest distance of 4.0 A, found for a guanidinium Czeta at the beta-turn, suggests N-H...O-P hydrogen bond formation. Torsion angle measurements of the two Arg's quantitatively confirm that the peptide adopts a beta-hairpin conformation in the lipid bilayer, and gel-phase 1H spin diffusion from water to the peptide indicates that PG-1 remains transmembrane in the gel phase of the membrane. For this transmembrane beta-hairpin peptide to have short (13)C-(31)P distances for multiple residues in the molecule, some phosphate groups must be embedded in the hydrophobic part of the membrane, with the local (31)P plane parallel to the beta-strand. This provides direct evidence for toroidal pores, where some lipid molecules change their orientation to merge the two monolayers. We propose that the driving force for this toroidal pore formation is guanidinium-phosphate complexation, where the cationic Arg residues drag the anionic phosphate groups along as they insert into the hydrophobic part of the membrane. This phosphate-mediated translocation of guanidinium ions may underlie the activity of other Arg-rich antimocrobial peptides and may be common among cationic membrane proteins.     

Accurate intermolecular ground state potential of the Ne-HCl van der Waals complex

From an accurate ground state intermolecular potential energy surface we evaluate the rovibrational spectrum of the Ne-HCl van der Waals complex. The intermolecular potential is obtained by fitting a considerable number of interaction energies obtained at the coupled cluster singles and doubles including connected triple excitations level and with the augmented correlation consistent polarized valence quintuple zeta basis set extended with a set of 3s3p2d1f1g midbond functions. This basis set is selected after a systematic basis set study carried out at geometries close to those of the three main surface stationary points. The surface is characterized by two linear minima, i.e. Ne-ClH and Ne-HCl, with distances from the Ne atom to the HCl center of mass of 3.398 and 3.833 angstroms, respectively; and binding energies of -65.10 and -66.85 cm(-1), respectively. These results agree well with the experimental data available in contrast to previous theoretical results. The rovibrational spectra calculated for the different isotopic species are also compared to the experiments.