Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: theory and application to membrane proteins

NMR anisotropic parameters such as dipolar couplings and chemical shifts are central to structure and orientation determination of aligned membrane proteins and liquid crystals. Among the separated local field experiments, the proton evolved local field (PELF) scheme is particularly suitable to measure dynamically averaged dipolar couplings and give information on local molecular motions. However, the PELF experiment requires the acquisition of several 2D datasets at different mixing times to optimize the sensitivity for the complete range of dipolar couplings of the resonances in the spectrum. Here, we propose a new PELF experiment that takes the advantage of the Hadamard encoding (HE) to obtain higher sensitivity for a broad range of dipolar couplings using a single 2D experiment. The HE scheme is obtained by selecting the spin operators with phase switching of hard pulses. This approach enables one to detect four spin operators, simultaneously, which can be processed into two 2D spectra covering a broader range of dipolar couplings. The advantages of the new approach are illustrated for a U-(15)N NAL single crystal and the U-(15)N labeled single-pass membrane protein sarcolipin reconstituted in oriented lipid bicelles. The HE-PELF scheme can be implemented in other multidimensional experiments to speed up the characterization of the structure and dynamics of oriented membrane proteins and liquid crystalline samples.     

Characterization of (1)H-(1)H distances in a uniformly (2)H,(15)N-labeled SH3 domain by MAS solid-state NMR spectroscopy (section sign)

In this communication, we demonstrate the feasibility of obtaining long-range (1)H-(1)H distance information by MAS solid-state NMR for a microcrystalline, uniformly (2)H,(15)N-labeled sample of a SH3 domain of chicken alpha-spectrin. The experiments yield NOESY-type spectra and rely on the favorable dispersion of the (15)N chemical shifts of the protein backbone. Perdeuteration of nonexchangeable sites is employed to simplify proton spin systems and to obtain multiple structural information. Two mixing schemes, (1)H-(1)H double quantum filtered Post-C7 and (1)H spin diffusion, are implemented to obtain quantitative (1)H-(1)H distance information. Post-C7 and spin diffusion cross-peak buildup rates are discussed for initial-rate fitting and in the framework of n = 0 rotational resonance (rotor driven spin diffusion), respectively. Different deuteration schemes were tested to find conditions where short-range (1)H-(1)H interactions are truncated (e.g., between H(N) and H(alpha)), but long-range interactions are retained (e.g., between H(N) and H(N)).     

PITANSEMA, a low-power PISEMA solid-state NMR experiment

A low radio frequency power polarization inversion spin exchange at the magic angle (PISEMA) pulse sequence is described for the measurement of heteronuclear dipolar couplings from solids. The method employs a time averaged nutation concept to significantly reduce the rf power required to spin-lock low gamma nuclear spins in PISEMA experiments. The efficacy of the 2D method is demonstrated on a single crystal of n-acetyl-L-(15)N-valyl-L-(15)N-leucine dipeptide to measure (1)H-(15)N dipolar couplings and a liquid crystal sample to measure (1)H-(13)C dipolar couplings.     

(1)H relaxation dispersion in solutions of nitroxide radicals: Effects of hyperfine interactions with (14)N and (15)N nuclei

(1)H relaxation dispersion of decalin and glycerol solutions of nitroxide radicals, 4-oxo-TEMPO-d(16)-(15)N and 4-oxo-TEMPO-d(16)-(14)N was measured in the frequency range of 10 kHz-20 MHz (for (1)H) using STELAR Field Cycling spectrometer. The purpose of the studies is to reveal how the spin dynamics of the free electron of the nitroxide radical affects the proton spin relaxation of the solvent molecules, depending on dynamical properties of the solvent. Combining the results for both solvents, the range of translational diffusion coefficients, 10(-9)-10(-11) m(2)∕s, was covered (these values refer to the relative diffusion of the solvent and solute molecules). The data were analyzed in terms of relaxation formulas including the isotropic part of the electron spin - nitrogen spin hyperfine coupling (for the case of (14)N and (15)N) and therefore valid for an arbitrary magnetic field. The influence of the hyperfine coupling on (1)H relaxation of solvent molecules depending on frequency and time-scale of the translational dynamics was discussed in detail. Special attention was given to the effect of isotope substitution ((14)N∕(15)N). In parallel, the influence of rotational dynamics on the inter-molecular (radical - solvent) electron spin - proton spin dipole-dipole coupling (which is the relaxation mechanism of solvent protons) was investigated. The rotational dynamics is of importance as the interacting spins are not placed in the molecular centers. It was demonstrated that the role of the isotropic hyperfine coupling increases for slower dynamics, but it is of importance already in the fast motion range (10(-9)m(2)∕s). The isotope effects is small, however clearly visible; the (1)H relaxation rate for the case of (15)N is larger (in the range of lower frequencies) than for (14)N. It was shown that when the diffusion coefficient decreases below 5 × 10(-11) m(2)∕s electron spin relaxation becomes of importance and its role becomes progressively more significant when the dynamics slows done. As far as the influence of the rotational dynamics is concerned, it was show that this process is of importance not only in the range of higher frequencies (like for diamagnetic solutions) but also at low and intermediate frequencies.     

15N solid-NMR and X-ray diffraction studies of N-confused porphyrins

Using (15)N high-resolution solid-state NMR and X-ray diffraction, the structure of N-confused porphyrin (NCP) in the solid state was studied. A 1D (15)N magic angle spinning (MAS) experiment and a 2D dipolar assisted rotational resonance (DARR) (15)N-(15)N spin exchange experiment of N-confused tetratolylporphyrin (Tol) crystallized from CH(2)Cl(2)/hexane indicate that Tol is the inner 3H-type tautomer and has two magnetically different molecules in the unit cell. Further, a FSLG-2 & 4macr; 2 (1)H-(15)N dipolar recoupling NMR measurement indicates no fast ring flipping motion which is consistent with the planar structure in the X-ray analysis. The planarity of Tol is ascribed to crystal packing enforced by pi-pi stacking and CH-pi interactions.     

Determination of natural abundance 15N-1H and 13C-1H dipolar couplings of molecules in a strongly orienting media using two-dimensional inverse experiments

NMR spectra of molecules oriented in liquid crystals provide homo- and heteronuclear dipolar couplings and thereby the geometry of the molecules. Several inequivalent dilute spins such as 13C and 15N coupled to protons form different coupled spin systems in their natural abundance and appear as satellites in the proton spectra. Identification of transitions belonging to each spin system is essential to determine heteronuclear dipolar couplings, which is a formidable task. In the present study, using 15N-1H and 13C-1H HSQC, and HMQC experiments we have selectively detected spectra of each rare spin coupled to protons. The 15N-1H and 13C-1H dipolar couplings have been determined in the natural abundance of 13C and 15N for the molecules pyrazine, pyrimidine and pyridazine oriented in a thermotropic liquid crystal.     

Geometry and spectral properties of the protonated homodimer of pyridine in the liquid and solid states. A combined NMR, X-ray diffraction and inelastic neutron scattering study

The structure and spectral signatures of the protonated homodimer of pyridine in its complex with a poorly coordinating anion have been studied in solution in CDF(3)/CDClF(2) down to 120 K and in a single crystal. In both phases, the hydrogen bond is asymmetric. In the solution, the proton is involved in a fast reversible transfer that determines the multiplicity of NMR signals and the sign of the primary H/D isotope effect of --0.95 ppm. The proton resonates at 21.73 ppm that is above any value reported in the past and is indicative of a very short hydrogen bond. By combining X-ray diffraction analysis with model computations, the position of the proton in the crystal has been defined as d(N-H) = 1.123 ? and d(H···N) = 1.532 ?. The same distances have been estimated using a (15)N NMR correlation. The frequency of the protonic out-of-plane bending mode is 822 cm(-1) in agreement with Novak's correlation.     

Dynamic NMR study of the mechanisms of double, triple, and quadruple proton and deuteron transfer in cyclic hydrogen bonded solids of pyrazole derivatives

Using dynamic solid state (15)N CPMAS NMR spectroscopy (CP = cross polarization, MAS = magic angle spinning), the kinetics of the degenerate intermolecular double and quadruple proton and deuteron transfers in the cyclic dimer of (15)N labeled polycrystalline 3,5-diphenyl-4-bromopyrazole (DPBrP) and in the cyclic tetramer of (15)N labeled polycrystalline 3,5-diphenylpyrazole (DPP) have been studied in a wide temperature range at different deuterium fractions in the mobile proton sites. Rate constants were measured on a millisecond time scale by line shape analysis of the doubly (15)N labeled compounds, and by magnetization transfer experiments on a second timescale of the singly (15)N labeled compounds in order to minimize the effects of proton-driven (15)N spin diffusion. For DPBrP the multiple kinetic HH/HD/DD isotope effects could be directly obtained. By contrast, four rate constants k(1) to k(4) were obtained for DPP at different deuterium fractions. Whereas k(1) corresponds to the rate constant k(HHHH) of the HHHH isotopolog, an appropriate kinetic reaction model was needed for the kinetic assignment of the other rate constants. Using the model described by Limbach, H. H.; Klein, O.; Lopez Del Amo, J. M.; Elguero, J. Z. Phys. Chem. 2004,218, 17, a concerted quadruple proton-transfer mechanism as well as a stepwise consecutive single transfer mechanism could be excluded. By contrast, using the kinetic assignment k(2) approximately k(3) approximately k(HHHD) approximately k(HDHD) and k(3) approximately k(HDDD) approximately k(DDDD), the results could be explained in terms of a two-step process involving a zwitterionic intermediate. In this mechanism, each reaction step involves the concerted transfer of two hydrons, giving rise to primary kinetic HH/HD/DD isotope effects, whereas the nontransferred hydrons only contribute small secondary effects, which are not resolved experimentally. By contrast, the multiple kinetic isotope effects of the double proton transfer in DPBrP and of the triple proton proton transfer in cyclic pyrazole trimers studied previously indicate concerted transfer processes. Thus, between n = 3 and 4 a switch of the reaction mechanism takes place. This switch is rationalized in terms of hydrogen bond compression effects associated with the multiple proton transfers. The Arrhenius curves of all processes are nonlinear and indicate tunneling processes at low temperatures. In a preliminary analysis, they are modeled in terms of the Bell-Limbach tunneling model.     

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.     

X–H rotational-echo double-resonance NMR for torsion angle determination of peptides

C–H and N–H rotational-echo double-resonance (REDOR) NMR is developed for determining torsion angles in peptides. The distance between an X spin such as ¹³C or ¹⁵N and a proton is measured by evolving the proton magnetization under REDOR-recoupled X–H dipolar interaction. The proton of interest is selected through its directly bonded heteronuclear spin Y. The sidechain torsion angle χ₁ is extracted from a ¹³Cβ-detected Hβ–N distance, while the backbone torsion angle φ is extracted from an ¹⁵N-detected H^N–C^′ distance. The approach is demonstrated on three model peptides with known crystal structures to illustrate its utility.     

Proton diffusion determination and dual structure model for nickel hydroxide based on potential step measurements on single spherical beads

Potential step measurement is carried out on single beads of spherical nickel hydroxide to determine the proton diffusion coefficient (D) and concentration of the effective proton vacancies (C). The semi-infinite diffusion equation for the initial stage and the finite diffusion equation for the long-term of the current response to potential step are used for deducing the D and C values. The diffusion coefficients deduced from short and long-term current responses are in the order of magnitude 10(-7) and 10(-10) cm2 s(-1), respectively. The sum of the effective proton vacancy concentrations associated with the two D values comes out to be equal within experimental error to the effective proton vacancy concentration converted from the released electricity during discharge. A dual structure model is proposed to interpret the above-mentioned findings, featuring densely packed grains within which proton diffusion is slow and an inter-grain matrix where proton diffusion is fast. With this model the huge difference (about 6 orders of magnitude) in D values reported in the literature as well as the controversy of the dependence of diffusion coefficient on the state of charge can be largely rationalized. This dual structure model is supported by SEM and AFM observations.     

Quantitative 15N NMR spectroscopy

Line intensities in ¹⁵N NMR spectra are strongly influenced by spin-lattice and spin–spin relaxation times, relaxation mechanisms and experimental conditions. Special care has to be taken in using ¹⁵N spectra for quantitative purposes. Quantitative aspects are discussed for the ¹⁵N NMR of molecules with different nitrogen functional groups and also mixtures of nitrogen-containing compounds. It is shown that, in general, quantitative data are obtainable from integration of ¹⁵N lines in proton decoupled ¹⁵N NMR spectra using NOE suppression. Addition of paramagnetic relaxation reagents (PARR) under controlled conditions is frequently needed to accomplish the experiment within reasonable time limits.     

Measurement of 15N-T1 relaxation rates in a perdeuterated protein by magic angle spinning solid-state nuclear magnetic resonance spectroscopy

In this paper, we present the measurement of (15)N-T(1) relaxation times in the solid state for a perdeuterated protein for which exchangeable protons are back substituted during recrystallization using a buffer which contains 10% H(2)O and 90% D(2)O. We find large variations of the (15)N relaxation time, even within the same beta sheet. By comparing (15)N-T(1) relaxation times measured for a protonated and a deuterated protein (using the above mentioned approach), we conclude that (1)H driven (15)N,(15)N spin diffusion has a significant impact on the absolute (15)N relaxation time in protonated proteins. This effect is important for a quantitative analysis of relaxation data in terms of molecular dynamics.     

NMR studies of ultrafast intramolecular proton tautomerism in crystalline and amorphous n,n'-diphenyl-6-aminofulvene-1-aldimine: solid-state, kinetic isotope, and tunneling effects

Using solid-state NMR spectroscopy, we have detected and characterized ultrafast intramolecular proton tautomerism in the N-H-N hydrogen bonds of solid N, N'-diphenyl-6-aminofulvene-1-aldimine ( I) on the microsecond-to-picosecond time scale. (15)N cross-polarization magic-angle-spinning NMR experiments using (1)H decoupling performed on polycrystalline I- (15)N 2 and the related compound N-phenyl- N'-(1,3,4-triazole)-6-aminofulvene-1-aldimine ( II) provided information about the thermodynamics of the tautomeric processes. We found that II forms only a single tautomer but that the gas-phase degeneracy of the two tautomers of I is lifted by solid-state interactions. Rate constants, including H/D kinetic isotope effects (KIEs), on the microsecond-to-picosecond time scale were obtained by measuring and analyzing the longitudinal (15)N and (2)H relaxation times of I- (15)N 2, I- (15)N 2- d 10, and I- (15)N 2- d 1 over a wide temperature range. In addition to the microcrystalline modification, a novel amorphous modification of I was found and studied. In this modification, proton transfer is much faster than in the crystalline form. For both modifications, we observed large H/D KIEs that were temperature-dependent at high temperatures and temperature-independent at low temperatures. These findings are interpreted in terms of a simple quasiclassical tunneling model proposed by Bell and modified by Limbach. We obtained evidence that a reorganization energy is necessary in order to compress the N-H-N hydrogen bond and achieve a molecular configuration in which the barrier for H transfer is reduced and tunneling or an over-barrier reaction can occur.     

Hydrophilic domain structure in polymer exchange membranes: Simulations of NMR spin diffusion experiments to address ability for model discrimination

We detail the development of a flexible simulation program (NMR_DIFFSIM) that solves the nuclear magnetic resonance (NMR) spin diffusion equation for arbitrary polymer architectures. The program was used to explore the proton (¹H) NMR spin diffusion behavior predicted for a range of geometrical models describing polymer exchange membranes. These results were also directly compared with the NMR spin diffusion behavior predicted for more complex domain structures obtained from molecular dynamics (MD) simulations. The numerical implementation and capabilities of NMR_DIFFSIM were demonstrated by evaluating the experimental NMR spin diffusion behavior for the hydrophilic domain structure in sulfonated Diels‐Alder Poly(Phenylene) (SDAPP) polymer membranes. The impact of morphology variations as a function of sulfonation and hydration level on the resulting NMR spin diffusion behavior were determined. These simulations allowed us to critically address the ability of NMR spin diffusion to discriminate between different structural models, and to highlight the extremely high fidelity experimental data required to accomplish this. A direct comparison of experimental double‐quantum‐filtered ¹H NMR spin diffusion in SDAPP membranes to the spin diffusion behavior predicted for MD‐proposed morphologies revealed excellent agreement, providing experimental support for the MD structures at low to moderate hydration levels. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 62–78     

Liquid crystal proton N.M.R. spectral analysis by numerical calculation

We offer an approximate method with which to calculate proton N.M.R. spectra generally caused by dipole-dipole interactions. The method consists of subdividing the spin system of the liquid crystal molecule into interacting blocks. The spectrum of each block is calculated exactly. The interaction between spins of different blocks is calculated approximately. The method is compared with the known methods using 4-n-pentyl-d₁₁-4'-cyanobiphenyl (5CB-d₁₁) and 4,4'-dimeth-oxyazoxybenzene (PAA) as examples.     

The proton uptake channel of bacteriorhodopsin as studied by a photoelectrochemical method

A series of the mutant proteins (D96N, D96N/D85N, D115N, L93T, T46V, V49A) where the residues are located at the cytoplasmic domain of bacteriorhodopsin (bR) were studied photoelectrochemically and their photocurrent response characteristics at the electrode/electrolyte interface were compared with those of the wild-type bR. While the wild-type bR of normal proton pumping activity yields symmetrical cathodic (positive) and anodic (negative) responses, corresponding to proton release and proton uptake, respectively, these mutants, with the exception of D115N, showed diminished amplitudes in the negative response. This indicates retardation of proton translocation from the cytoplasmic surface to the retinal Schiff base. The mutation that gave the strongest influence on the negative response was D96N while moderate influence was obtained with L93T, T46V, and V49A. These results suggest that residues other than D96 also participate in the cytoplasmic proton uptake channel, either by interacting with D96 directly or by forming a hydrogen-bonded network with water molecules. The D96N/D85N double mutant yielded little response at neutral pH, but the response was partially recovered by addition of azide, while it was fully recovered in the single mutant D96N. The D115N mutant showed the response profile that closely resembles the wild-type, indicating that D115 is not crucially involved in the event of proton transfer relay at the cytoplasmic region. It was also found that every mutant in this study releases protons prior to uptake at the other membrane surface, as does the wild-type.     

Dynamic nuclear polarization of amyloidogenic peptide nanocrystals: GNNQQNY, a core segment of the yeast prion protein Sup35p

Dynamic nuclear polarization (DNP) permits a approximately 10(2)-10(3) enhancement of the nuclear spin polarization and therefore increases sensitivity in nuclear magnetic resonance (NMR) experiments. Here, we demonstrate the efficient transfer of DNP-enhanced (1)H polarization from an aqueous, radical-containing solvent matrix into peptide crystals via (1)H-(1)H spin diffusion across the matrix-crystal interface. The samples consist of nanocrystals of the amyloid-forming peptide GNNQQNY(7-13), derived from the yeast prion protein Sup35p, dispersed in a glycerol-water matrix containing a biradical polarizing agent, TOTAPOL. These crystals have an average width of 100-200 nm, and their known crystal structure suggests that the size of the biradical precludes its penetration into the crystal lattice; therefore, intimate contact of the molecules in the nanocrystal core with the polarizing agent is unlikely. This is supported by the observed differences between the time-dependent growth of the enhanced polarization in the solvent versus the nanocrystals. Nevertheless, DNP-enhanced magic-angle spinning (MAS) spectra recorded at 5 T and 90 K exhibit an average signal enhancement epsilon approximately 120. This is slightly lower than the DNP enhancement of the solvent mixture surrounding the crystals (epsilon approximately 160), and we show that it is consistent with spin diffusion across the solvent-matrix interface. In particular, we correlate the expected DNP enhancement to several properties of the sample, such as crystal size, the nuclear T(1), and the average (1)H-(1)H spin diffusion constant. The enhanced (1)H polarization was subsequently transferred to (13)C and (15)N via cross-polarization, and allowed rapid acquisition of two-dimensional (13)C-(13)C correlation data.     

Lamellar structure in poly(ala-gly) determined by solid-state NMR and statistical mechanical calculations

Lamellar structure of poly(Ala-Gly) or (AG)n in the solid was examined using 13C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1-13C]Gly14[1-13C]Ala15- and [1-13C]Gly18[1-13C]Ala19 of (AG)15 were examined by two-dimensional (2D) 13C spin diffusion NMR in the solid state. In addition five doubly labeled [15N,13C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3-13C] in each of the successive Ala residues were utilized for REDOR and 13C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted beta-turns with a large distribution of the torsion angles and antiparallel beta-sheets. The relative proportion of the distorted beta-turn form was evaluated by examination of 13C CP/MAS NMR spectra of [3-13C]Ala-(AG)15. In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13C and 15N nuclei which were also interpreted in terms of a combination of beta-sheets and beta-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cbeta 13C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.     

NMR detection of pH-dependent histidine-water proton exchange reveals the conduction mechanism of a transmembrane proton channel

The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole-imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, (15)N NMR spectra show fast exchange of the imidazole (15)N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the (15)N T(2) relaxation times and simulated them for chemical-shift exchange and fluctuating N-H dipolar fields under (1)H decoupling and MAS. The exchange rate is 4.5 × 10(5) s(-1) for Nδ1 and 1.0 × 10(5) s(-1) for Nε2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four pK(a) values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these pK(a)'s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal helical structure coexists with a broadly distributed ideal helical conformation without interchange on the sub-10 ms time scale, thus excluding the transporter model in the viral membrane. These data support the shuttle mechanism of proton conduction, whose essential steps involve His-water proton exchange facilitated by imidazole ring reorientations.