High resolution 2D 1H-13C correlation of cholesterol in model membrane

High resolution 2D NMR MAS spectra of liposomes, in particular 1H-13C chemical shifts correlations have been obtained on fluid lipid bilayers made of pure phospholipids for several years. We have investigated herein the possibility to obtain high resolution 2D MAS spectra of cholesterol embedded in membranes, i.e. on a rigid molecule whose dynamics is characterized mainly by axial diffusion without internal segmental mobility. The efficiency of various pulse sequences for heteronuclear HETCOR has been compared in terms of resolution, sensitivity and selectivity, using either cross polarization or INEPT for coherence transfer, and with or without MREV-8 homonuclear decoupling during t1. At moderately high spinning speed (9 kHz), a similar resolution is obtained in all cases (0.2 ppm for 1H(3,4), 0.15 ppm for 13C(3,4) cholesterol resonances), while sensitivity increases in the order: INEPT < CP(x4) < CP + MREV. At reduced spinning speed (5 kHz), the homonuclear dipolar coupling between the two geminal protons attached to C(4) gives rise to spinning sidebands from which one can estimate a H-H dipolar coupling of 10 kHz which is in good agreement with the known dynamics of cholesterol in membranes.     

Selective dephasing of OH and NH proton magnetization based on (1)H chemical-shift anisotropy recoupling

A method for selectively suppressing the signals of OH and NH protons in (1)H combined rotation and multiple-pulse spectroscopy (CRAMPS) and in (1)H-(13)C heteronuclear correlation (HETCOR) solid-state NMR spectra is presented. It permits distinction of overlapping CH and OH/NH proton signals, based on the selective dephasing of the magnetization of OH and NH protons by their relatively large (1)H chemical-shift anisotropies. For NH protons, the (14)N-(1)H dipolar coupling also contributes significantly to this dephasing. The dephasing is achieved by a new combination of heteronuclear recoupling of these anisotropies with (1)H homonuclear dipolar decoupling. Since the 180 degrees pulses traditionally used for heteronuclear dipolar and chemical-shift anisotropy recoupling would result in undesirable homonuclear dephasing of proton magnetization, instead the necessary inversion of the chemical-shift Hamiltonian every half rotation period is achieved by inverting the phases of all the pulses in the HW8 multiple-pulse sequence. In the HETCOR experiments, carefully timed (13)C 180 degrees pulses remove the strong dipolar coupling to the nearby (13)C spin. The suppression of NH and OH peaks is demonstrated on crystalline model compounds. The technique in combination with HETCOR NMR is applied to identify the CONH and NH-CH groups in chitin and to distinguish NH and aromatic proton peaks in a peat humin.     

High-resolution heteronuclear correlation spectroscopy in solid state NMR of aligned samples

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

1H assisted 13C/15N heteronuclear correlation spectroscopy in oriented sample solid-state NMR of single crystal and magnetically aligned samples

(1)H-irradiation under mismatched Hartmann-Hahn conditions provides an alternative mechanism for carrying out (15)N/(13)C transfers in triple-resonance heteronuclear correlation spectroscopy (HETCOR) on stationary samples of single crystals and aligned samples of biopolymers, which improve the efficiency especially when the direct (15)N-(13)C dipolar couplings are small. In many cases, the sensitivity is improved by taking advantage of the (13)C(α) labeled sites in peptides and proteins with (13)C detection. The similarities between experimental and simulated spectra demonstrate the validity of the recoupling mechanism and identify the potential for applying these experiments to virus particles or membrane proteins in phospholipid bilayers; however, further development is needed in order to derive quantitative distance and angular constraints from these measurements.     

Proton-detected separated local field spectroscopy

PISEMO, a separated local field experiment that can be performed with either direct (15)N (or (13)C) detection or indirect (1)H detection, is demonstrated on a single crystal of a model peptide. The (1)H signals modulated by (1)H-(15)N heteronuclear dipole-dipole couplings are observed stroboscopically in the windows of the multiple-pulse sequence used to attenuate (1)H-(1)H homonuclear dipole-dipole couplings. (1)H-detection yields spectra with about 2.5 times the signal to noise ratio observed with (15)N-detection under equivalent conditions. Resolution in both the (15)N chemical shift and (1)H-(15)N heteronuclear dipole-dipole coupling dimensions is similar to that observed with PISEMA, however, since only on-resonance pulses are utilized, the bandwidth is better.     

Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR

We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and high redundancy in the primary sequence. We implemented this approach in both heteronuclear (15)N-(13)C(α) and homonuclear (13)C-(13)C dipolar assisted rotational resonance (DARR) correlation experiments. We demonstrate its efficacy for the membrane protein phospholamban reconstituted in fluid PC/PE/PA lipid bilayers. The main advantage of this method is to discriminate overlapped (13)C(α) resonances by strategically labeling the preceding residue. This method is highly complementary to (13)C(i-1)(')-(15)N(i)-(13)C(i)(α) and (13)C(i-1)(α)-(15)N(i-1)-(13)C(i)(') experiments to distinguish inter-residue spin systems at a minimal cost to signal-to-noise.     

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Clean MAS observation of 13C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13C-15N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13C carbonyl and its directly bonded 15N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic- and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U-13C, 15N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.     

A comparison of NCO and NCA transfer methods for biological solid-state NMR spectroscopy

Three different techniques (adiabatic passage Hartman-Hahn cross-polarization, optimal control designed pulses, and EXPORT) are compared for transferring (15)N magnetization to (13)C in solid-state NMR experiments under magic-angle-spinning conditions. We demonstrate that, in comparison to adiabatic passage Hartman-Hahn cross-polarization, optimal control transfer pulses achieve similar or better transfer efficiencies for uniformly-(13)C,(15)N labeled samples and are generally superior for samples with non-uniform labeling schemes (such as 1,3- and 2-(13)C glycerol labeling). In addition, the optimal control pulses typically use substantially lower average RF field strengths and are more robust with respect to experimental variation and RF inhomogeneity. Consequently, they are better suited for demanding samples.     

Triple resonance experiments for aligned sample solid-state NMR of (13)C and (15)N labeled proteins

Initial steps in the development of a suite of triple-resonance (1)H/(13)C/(15)N solid-state NMR experiments applicable to aligned samples of (13)C and (15)N labeled proteins are described. The experiments take advantage of the opportunities for (13)C detection without the need for homonuclear (13)C/(13)C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are approximately 20% randomly labeled with (13)C in all backbone and side chain carbon sites and approximately 100% uniformly (15)N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are (13)C labeled at only the alpha-carbon and (15)N labeled at the amide nitrogen of a few residues. The requirement for homonuclear (13)C/(13)C decoupling while detecting (13)C signals is avoided in the first case because of the low probability of any two (13)C nuclei being bonded to each other; in the second case, the labeled (13)C(alpha) sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the (13)C chemical shift and (1)H-(13)C and (15)N-(13)C heteronuclear dipolar coupling frequencies associated with the (13)C(alpha) and (13)C' backbone sites, which provide orientation constraints complementary to those derived from the (15)N labeled amide backbone sites. (13)C/(13)C spin-exchange experiments identify proximate carbon sites. The ability to measure (13)C-(15)N dipolar coupling frequencies and correlate (13)C and (15)N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the (13)C chemical shift and (1)H-(13)C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.     

A solid-state NMR application of the anomeric effect in carbohydrates: galactosamine, glucosamine, and N-acetyl-glucosamine

Simple 2D 13C/15N heteronuclear correlation solid-state NMR spectroscopy was implemented to resolve the 15N resonances of the alpha and beta anomers of three amino monosaccharides: galactosamine (GalN), glucosamine hydrochloride (GlcN), and N-acetyl-glucosamine (GlcNAc) labeled specifically with 13C1/15N spin pairs. Although the 15N resonances could not be distinguished in normal 1D spectra, they were well resolved in 2D double CP/MAS correlation spectra by taking advantage of the 13C spectral resolution. The alpha and beta resonances shifted apart by 3-5 ppm in their 13C chemical shifts, and differed by 1-2 ppm in the extended 15N dimension. Aside from this, the detection of other 13C/15N correlations over short distances was also achieved arising from the C2, C3 and CO carbons present in natural abundance. 2D double CP/MAS chemical shift correlation NMR spectroscopy is a simple and powerful technique to characterize the anomeric effect of amino monosaccharides. Applications of the 2D method reveal well-resolved 15N and 13C chemical shifts might be useful for structural determination on carbohydrates of biological significance, such as glycopeptide or glycolipids.     

Coherence selection in double CP MAS NMR spectroscopy

Applications of double cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy, via (1)H/(15)N and then (15)N/(13)C coherence transfers, for (13)C coherence selection are demonstrated on a (15)N/(13)C-labeled N-acetyl-glucosamine (GlcNAc) compound. The (15)N/(13)C coherence transfer is very sensitive to the settings of the experimental parameters. To resolve explicitly these parameter dependences, we have systematically monitored the (13)C{(15)N/(1)H} signal as a function of the rf field strength and the MAS frequency. The data reveal that the zero-quantum coherence transfer, with which the (13)C effective rf field is larger than that of the (15)N by the spinning frequency, would give better signal sensitivity. We demonstrate in one- and two-dimensional double CP experiments that spectral editing can be achieved by tailoring the experimental parameters, such as the rf field strengths and/or the MAS frequency.     

Determination of the backbone torsion psi angle by tensor correlation of chemical shift anisotropy and heteronuclear dipole-dipole interaction

We demonstrate that the backbone torsion psi angle of a uniformly labeled residue can be determined accurately by correlating the chemical shift anisotropy of the carbonyl carbon and the 13C-1H heteronuclear dipole-dipole interaction of the alpha carbon. To obtain the highest sensitivity for the psi angle determination, the following conditions are desired: (i) the recoupling pulse sequences for the CSA and the heteronuclear dipolar interactions are gamma encoded, in which the spatial parts of m=2 are selected; (ii) the homonuclear polarization transfer is based on the scalar spin-spin coupling. Experimental data were obtained for [U-13C, 15N]-alanine and N-acetyl-[U-13C, 15N]-d,l-valine under magic-angle spinning at 25kHz. Only three data points are required for the measurements and the dihedral angles determined are in excellent agreement with the diffraction data.     

SPINAL modulated decoupling in high field double- and triple-resonance solid-state NMR experiments on stationary samples

Continuous wave irradiation has limited bandwidth for heteronuclear 1H decoupling at high fields and for 13C decoupling in 1H/13C/15N triple-resonance experiments. SPINAL-16 modulation is shown to improve the efficiency of 1H and 13C heteronuclear decoupling on single crystals of peptides and on magnetically aligned samples of membrane proteins in bicelles, which is of particular importance because aqueous samples of biomolecules are lossy at high fields, which limits the strengths of the RF fields that can be applied.     

3D NMR spectroscopy for resonance assignment and structure elucidation of proteins under MAS: novel pulse schemes and sensitivity considerations

Two types of 3D MAS NMR experiments are introduced, which combine standard (NC,CC) transfer schemes with (1H,1H) mixing to simultaneously detect connectivities and structural constraints of uniformly 15N,13C-labeled proteins with high spectral resolution. The homonuclear CCHHC and CCC experiments are recorded with one double-quantum evolution dimension in order to avoid a cubic diagonal in the spectrum. Depending on the second transfer step, spin systems or proton-proton contacts can be determined with reduced spectral overlap. The heteronuclear NHHCC experiment encodes NH-HC proton-proton interactions, which are indicative for the backbone conformation of the protein. The third dimension facilitates the identification of the amino acid spin system. Experimental results on U-[15N,13C]valine and U-[15N,13C]ubiquitin demonstrate their usefulness for resonance assignments and for the determination of structural constraints. Furthermore, we give a detailed analysis of alternative multidimensional sampling schemes and their effect on sensitivity and resolution.     

Determination of chemical shift anisotropies of unresolved carbonyl sites by C-alpha detection under magic-angle spinning

We demonstrate that the static powder pattern line shape of chemical shift anisotropy (CSA) can be obtained for unresolved carbonyl sites of polypeptides under magic-angle spinning. The CSA interaction is first recoupled at the carbonyl site. The phase factors associated with the CSA recoupling are then transferred to the adjacent alpha carbon by an isotropic polarization transfer based on scalar spin-spin coupling. Because alpha carbons of polypeptides are usually better resolved, we can then obtain the CSA static powder pattern line shapes of the carbonyl sites after Fourier transformation in the second dimension. We validate our approach experimentally by measurements on [U-(13)C, (15)N]-l-alanine, [U-(13)C, (15)N]-l-valine and prion fibrils with uniform (13)C and (15)N labels on selected residues.     

A new two-dimensional pulse sequence for T(2)* measurements of protons in (13)C isotopomers

A new two-dimensional pulse sequence for T(2)* measurement of protons directly coupled to (13)C spins is proposed. The sequence measures the tranverse relaxation time of heteronuclear proton single-quantum coherence under conditions of free precession and is therefore well suited to evaluate relaxation losses of proton magnetization during preparation delays of heteronuclear pulse experiments in analytical NMR. The relevant part of the pulse sequence can be inserted as a "building block" into any direct or inverse detecting H,C correlation pulse sequence if proton spin-spin relaxation is to be investigated. In this contribution, the building block is inserted into a HETCOR as well as into a HMQC pulse sequence. Experimental results for the HETCOR-based sequence are given.     

Exploring Chromophore-Binding Pocket: High-Resolution Solid-State 1H–13C Interfacial Correlation NMR Spectra with Windowed PMLG Scheme

High-resolution two-dimensional (2D) ¹H–¹³C heteronuclear correlation spectra are recorded for selective observation of interfacial 3–5.5 ? contacts of the uniformly ¹³C-labeled phycocyanobilin (PCB) chromophore with its unlabeled binding pocket. The experiment is based on a medium- and long-distance heteronuclear correlation (MELODI–HETCOR) method. For improving ¹H spectral resolution, a windowed phase-modulated Lee–Goldburg (wPMLG) decoupling scheme is applied during the t ₁ evolution period. Our approach allows for identification of chromophore–protein interactions, in particular for elucidation of the hydrogen-bonding networks and charge distributions within the chromophore-binding pocket. The resulting pulse sequence is tested on the cyanobacterial (Cph1) phytochrome sensory module (residues 1–514, Cph1Δ2) containing uniformly ¹³C- and ¹⁵N-labeled PCB chromophore (u-[¹³C,¹⁵N]-PCB-Cph1Δ2) at 17.6 T.     

Sensitivity considerations in polarization transfer and filtering using dipole-dipole couplings: implications for biomineral systems

The robustness and sensitivities of different polarization-transfer methods that exploit heteronuclear dipole-dipole couplings are compared for a series of heterogeneous solid systems, including polycrystalline tetrakis(trimethylsilyl)silane (TKS), adamantane, a physical mixture of doubly (13)C,(15)N-enriched and singly (13)C-enriched polycrystalline glycine, and a powder sample of siliceous marine diatoms, Thalossiosira pseudonana. The methods were analyzed according to their respective frequency-matching spectra or resultant signal intensities. For a series of (13)C{(1)H} cross-polarization experiments, adiabatic passage Hartmann-Hahn cross-polarization (APHH-CP) was shown to have several advantages over other methods, including Hartmann-Hahn cross-polarization (HHCP), variable-amplitude cross-polarization (VACP), and ramped-amplitude cross-polarization (RACP). For X-Y systems, such as (13)C{(15)N}, high and comparable sensitivities were obtained by using APHH-CP with Lee-Goldburg decoupling or by using the transferred-echo double resonance (TEDOR) experiment. The findings were applied to multinuclear (1)H, (13)C, (15)N, and (29)Si CP MAS characterization of a powder diatom sample, a challenging inorganic-organic hybrid solid that places high demands on NMR signal sensitivity.     

Three-dimensional 13C shift/1H-15N coupling/15N shift solid-state NMR correlation spectroscopy.

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly 13C- and 15N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from 1H to 13C and from 13C to 15N with flip-flop (phase and frequency switched) Lee-Goldburg irradiation for both 13C homonuclear decoupling and 1H-15N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields 13C shift/1H-15N coupling/15N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate 13C and 15N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly 13C- and 15N-labeled proteins.     

Pursuing structure in microcrystalline solids with independent molecules in the unit cell using 1H-13C correlation data

The (1)H-(13)C solid-state NMR heteronuclear correlation (HETCOR) experiment is demonstrated to provide shift assignments in certain powders that have two or more structurally independent molecules in the unit cell (i.e. multiple molecules per asymmetric unit). Although this class of solids is often difficult to characterize using other methods, HETCOR provides both the conventional assignment of shifts to molecular positions and associates many resonances with specific molecules in the asymmetric unit. Such assignments facilitate conformational characterization of the individual molecules of the asymmetric unit and the first such characterization solely from solid-state NMR data is described. HETCOR offers advantages in sensitivity over prior methods that assign resonances in the asymmetric unit by (13)C-(13)C correlations and therefore allows shorter average analysis times in natural abundance materials. The (1)H-(13)C analysis is demonstrated first on materials with known shift assignments from INADEQUATE data (santonin and Ca(OAc)(2) phase I) to verify the technique and subsequently is extended to a pair of unknown solids: (+)-catechin and Ca(OAc)(2) phase II. Sufficient sensitivity and resolution is achieved in the spectra to provide assignments to one of the specific molecules of the asymmetric unit at over 54% of the sites.