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.     

Two- and Three-Dimensional H/C PISEMA Experiments and Their Application to Backbone and Side Chain Sites of Amino Acids and Peptides

Two-dimensional ¹H/¹³C polarization inversion spin exchange at the magic angle experiments were applied to single crystal samples of amino acids to demonstrate their potential utility on oriented samples of peptides and proteins. High resolution is achieved and structural information obtained on backbone and side chain sites from these spectra. A triple-resonance experiment that correlates the ¹H–¹³C_α dipolar coupling frequency with the chemical shift frequencies of the α-carbon, as well as the directly bonded amide ¹⁵N site, is also demonstrated. In this experiment the large ¹H–¹³C_α heteronuclear dipolar interaction provides an independent frequency dimension that significantly improves the resolution among overlapping ¹³C resonances of oriented polypeptides, while simultaneously providing measurements of the ¹³C_α chemical shift, ¹H–¹³C dipolar coupling, and ¹⁵N chemical shift frequencies and angular restraints for backbone structure determination.     

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.     

HCCCH Experiment for Through-Bond Correlation of Thymine Resonances inC-Labeled DNA Oligonucleotides

Application of heteronuclear magnetic resonance pulse methods to¹³C,¹⁵N-labeled nucleic acids is important for the accurate structure determination of larger RNA and DNA oligonucleotides and protein–nucleic acid complexes. These methods have been applied primarily to RNA, due to the availability of labeled samples. The two major differences between DNA and RNA are at the C2′ of the ribose and deoxyribose and the additional methyl group on thymine versus uracil. We have enzymatically synthesized a¹³C,¹⁵N-labeled 32 base DNA oligonucleotide that folds to form an intramolecular triplex. We present two- and three-dimensional versions of a new HCCCH–TOCSY experiment that provides intraresidue correlation between the thymine H6 and methyl resonances via the intervening carbons (H6–C6–C5–Cme–Hme).     

Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins. 1990

Four new and complementary three-dimensional triple-resonance experiments are described for obtaining complete backbone ¹H, ¹³C, and ¹⁵N resonance assignments of proteins uniformly enriched with ¹³C and ¹⁵N. The new methods all rely on ¹H detection and use multiple magnetization transfers through well-resolved one-bond J couplings. Therefore, the 3D experiments are sensitive and permit relatively rapid recording of 3D spectra (l–2 days) for protein concentrations on the order of 1 mM. One experiment (HNCO) correlates the amide ¹H and ¹⁵N shifts with the ¹³C shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amide ¹H and ¹⁵N shifts with the Cα chemical shift. This experiment often also provides a weak correlation between the amide NH and ¹⁵N resonances of one amino acid and the Ca resonance of the preceding amino acid. A third experiment (HCACO) correlates the Hα and Cα shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA(CO)N, correlates Ha and Cal resonances of one residue with the ¹⁵N frequency of the succeeding residue. The principles of these experiments are described in terms of the operator formalism. To optimize spectral resolution, special attention is paid to removal of undesired J splittings in the 3D spectra. Technical details regarding the implementation of these triple-resonance experiments on a commercial spectrometer are also provided. The experiments are demonstrated for the protein calmodulin (16.7 kDa).     

Suppression of artifacts induced by homonuclear decoupling in amino-acid-type edited methyl H–C correlation experiments

A detailed theoretical and experimental analysis of the artifacts induced by homonuclear band-selective decoupling during CT frequency labeling is presented. The effects are discussed in the context of an amino-acid-type editing filter implemented in ¹H–¹³C CT-HSQC experiments of methyl groups in proteins. It is shown that both Bloch–Siegert shifts and modulation sidebands are efficiently suppressed by using additional off-resonance decoupling as proposed by Zhang and Gorenstein [J. Magn. Reson. 132 (1998) 81], and appropriate adjustment of a set of pulse sequence parameters. The theoretical predictions are confirmed by experiments performed on ¹³C-labeled protein samples, yielding artifact-free amino-acid-type edited methyl spectra.     

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.     

1H detected 1H,15N correlation spectroscopy in rotating solids

We describe new correlation experiments suitable for determining long-range 1H-1H distances in 2H,15N-labeled peptides and proteins. The approach uses perdeuteration together with back substitution of exchangeable protons during sample preparation as a means of attenuating the strong 1H-1H dipolar couplings that broaden 1H magic angle spinning (MAS) spectra of solids. In the approach described here, we retain 100% of the 1H sensitivity by labeling and detecting all exchangeable sites. This is in contrast to homonuclear multiple pulse decoupling sequences that are applied during detection and that compromise sensitivity because of the requirement of sampling between pulses. As a result 1H detection provides a gain in sensitivity of >5 compared to the 15N detected version of the experiment (at a MAS frequency of 13.5kHz). The pulse schemes make use of the favorable dispersion of the amide 15Ns resonances in the protein backbone. The experiments are demonstrated on a sample of the uniformly 2H,15N-labeled dipeptide N-Ac-Val-Leu-OH and are analogous to the solution-state suite of HSQC-NOESY experiments. In this compound the 1H amide linewidths at 750MHz vary from approximately 0.67 ppm at omega(r)/2pi approximately 5kHz to approximately 0.20 ppm at omega(r)/2pi approximately 30kHz, indicating that useful resolution is available in the 1H spectrum via this approach. Since the experiments circumvent the problem of dipolar truncation in the 1H-1H spin system, they should make it possible to measure long-range distances in a uniformly labeled environment. Thus, we expect the experiments to be useful in constraining the global fold of a protein.     

Separating Structure and Dynamics in CSA/DD Cross-Correlated Relaxation: A Case Study on Trehalose and Ubiquitin

We present two new sensitivity enhanced gradient NMR experiments for measuring interference effects between chemical shift anisotropy (CSA) and dipolar coupling interactions in a scalar coupled two-spin system in both the laboratory and rotating frames. We apply these methods for quantitative measurement of longitudinal and transverse cross-correlation rates involving interference of ¹³C CSA and ¹³C–¹H dipolar coupling in a disaccharide, α,α- -trehalose, at natural abundance of ¹³C as well as interference of amide ¹⁵N CSA and ¹⁵N–¹H dipolar coupling in uniformly ¹⁵N-labeled ubiquitin. We demonstrate that the standard heteronuclear T₁, T₂, and steady-state NOE autocorrelation experiments augmented by cross-correlation measurements provide sufficient experimental data to quantitatively separate the structural and dynamic contributions to these relaxation rates when the simplifying assumptions of isotropic overall tumbling and an axially symmetric chemical shift tensor are valid.     

The roles of homonuclear line narrowing and the1H amide chemical shift tensor in structure determination of proteins by solid-state NMR spectroscopy

The seminal contributions of Ulrich Haeberlen to homonuclear line narrowing and the determination of¹H chemical shift tensors are crucial for protein structure determination by solid-state nuclear magnetic resonance spectroscopy. The¹H chemical shift is particularly important in spectra obtained on oriented samples of membrane proteins as a mechanism for providing dispersion among resonances that are not resolved with the¹H-¹⁵N dipolar coupling and¹⁵N chemical shift frequencies. This is demonstrated with three-dimensional experiments on uniformly¹⁵N-labeled samples of Magainin antibiotic peptide and the protein Vpu from HIV-1 in oriented lipid bilayers. These experiments enable resonances in two-dimensional¹H-¹⁵N dipolar coupling/¹⁵N chemical shift planes separated by¹H chemical shift frequencies to be resolved and analyzed. These three-dimensional spectra are compared to one-dimensional spectra of full-length Vpu, the cytoplasmic domain of Vpu, and Magainin, as well as to two-dimensional spectra of fd coat protein and Colicin El polypeptide. The¹H amide chemical shift tensor provides valuable structural information, and this is demonstrated with its contributions to orientational restrictions to one of the in-plane helical residues of Magainin.     

Experimental access to HSQC spectra decoupled in all frequency dimensions

A new operator called RESET “Reducing nuclEar Spin multiplicitiEs to singuleTs” is presented to acquire broadband proton decoupled proton spectra in one and two dimensions. Basically, the homonuclear decoupling is achieved through the application of bilinear rotation pulses and delays. A [BIRD]^r,x pulse building block is used to selectively invert all proton magnetization remotely attached to ¹³C isotopes, which is equivalent to a scalar J decoupling of the protons directly attached to ¹³C from all other protons in the spin system. In conjunction with an appropriate data processing technique pure shift proton spectra are obtained. For this purpose, the concept of constant time acquisition in the observe dimension is exploited. Both ideas were merged together producing superior HSQC based pseudo 3D pulse sequences. The resulting HSQC spectra show cross peaks with collapsed multiplet structures and singlet responses for the proton chemical shift frequencies. An unambiguous assignment of signals from overcrowded spectra becomes much easier. Finally, the recently introduced SHARC technique is exploited to enhance the capability of the scalar J decoupling method. A significant reduction of the total measurement time is achieved. The time is saved by reducing the number of ¹³C chemical shift evolution increments and working with superimposed narrow spectral bandwidths in the ¹³C indirect domain.     

Structural analysis of uniformly C-labelled solids from selective angle measurements at rotational resonance

We demonstrate that individual H–C–C–H torsional angles in uniformly labelled organic solids can be estimated by selective excitation of ¹³C double-quantum coherences under magic-angle spinning at rotational resonance. By adapting a straightforward one-dimensional experiment described earlier [T. Karlsson, M. Eden, H. Luhman, M.H. Levitt, J. Magn. Reson. 145 (2000) 95–107], a double-quantum filtered spectrum selective for Cα and Cβ of uniformly labelled l-[¹³C,¹⁵N]valine is obtained with 25% efficiency. The evolution of Cα–Cβ double-quantum coherence under the influence of the dipolar fields of bonded protons is monitored to provide a value of the Hα–Cα–Cβ–Hβ torsional angle that is consistent with the crystal structure. In addition, double-quantum filtration selective for C6 and C1′ of uniformly labelled [¹³C,¹⁵N]uridine is achieved with 12% efficiency for a ¹³C–¹³C distance of 2.5 Å, yielding a reliable estimate of the C6–H and C1′–H projection angle defining the relative orientations of the nucleoside pyrimidine and ribose rings. This procedure will be useful, in favourable cases, for structural analysis of fully labelled small molecules such as receptor ligands that are not readily synthesised with labels placed selectively at structurally diagnostic sites.     

Optimization of Three-Dimensional TROSY-Type HCCH NMR Correlation of Aromatic H–C Groups in Proteins

Improved methods for three-dimensional TROSY-Type HCCH correlation involving protons of negligible CSA are presented. The TROSY approach differs from the conventional approach of heteronuclear decoupling in evolution and detection periods by not mixing fast and slowly relaxing coherences and usually suppressing the former. Pervushin et al. (J. Am. Chem. Soc. 120, 6394–6400 (1998)) have proposed a 3D TROSY-type HCCH experiment where the TROSY approach is applied only in one of the ¹³C dimensions. A new pulse sequence applying the TROSY approach in both indirect dimensions is advantageous when the TROSY effect of the carbons is large or when a relatively high resolution is required. For lower resolutions or moderate TROSY effects we show that it is possible to combine the best of both worlds, namely to suppress heteronuclear couplings without mixing fast and slowly relaxing coherences while at the same time superimpose the two components and thus have both contribute to the detected signal. That is possible using the novel technique of Spin-State-Selective Time-Proportional Phase Incrementation (S³ TPPI). The new 3D S³ TPPI TROSY HCCH method is demonstrated on a ¹³C,¹⁵N-labeled protein sample, RAP 18–112 (N-terminal domain of α₂-macroglobulin receptor associated protein), at 750 MHz and average sensitivity enhancements of 10% are obtained for the cross peaks in comparison to methods based on conventional decoupling on one of the carbons or on TROSY on both carbons.     

N Chemical Shift Tensor Magnitude and Orientation in the Molecular Frame of Uracil Determined via MAS NMR

The potential of heteronuclear MAS NMR spectroscopy for the characterization of ¹⁵N chemical shift (CS) tensors in multiply labeled systems has been illustrated, in one of the first studies of this type, by a measurement of the chemical shift tensor magnitude and orientation in the molecular frame for the two ¹⁵N sites of uracil. Employing polycrystalline samples of ¹⁵N₂ and 2-¹³C,¹⁵N₂-labeled uracil, we have measured, via ¹⁵N–¹³C REDOR and ¹⁵N–¹H dipolar-shift experiments, the polar and azimuthal angles (θ, ψ) of orientation of the ¹⁵N–¹³C and ¹⁵N–¹H dipolar vectors in the ¹⁵N CS tensor frame. The (θ_NC, ψ_NC) angles are determined to be (92 ± 10°, 100 ± 5°) and (132 ± 3°, 88 ± 10°) for the N1 and N3 sites, respectively. Similarly, (θ_NH, ψ_NH) are found to be (15 ± 5°, −80 ± 10°) and (15 ± 5°, 90 ± 10°) for the N1 and N3 sites, respectively. These results obtained based only on MAS NMR measurements have been compared with the data reported in the literature.     

C Chemical Shift and C–N Dipolar Coupling Tensors Determined by C Rotary Resonance Solid-State NMR

This work explores the utility of simple rotary resonance experiments for the determination of the magnitude and orientation of ¹³C chemical shift tensors relative to one or more ¹³C–¹⁴N internuclear axes from ¹³C magic-angle-spinning NMR experiments. The experiment relies on simultaneous recoupling of the anisotropic ¹³C chemical shift and ¹³C–¹⁴N dipole–dipole coupling interactions using 2D rotary resonance NMR with RF irradiation on the ¹³C spins only. The method is demonstrated by experiments and numerical simulations for the ¹³C^α spins in powder samples of -alanine and glycine with ¹³C in natural abundance. To investigate the potential of the experiment for determination of relative/absolute tensor orientations and backbone dihedral angles in peptides, the influence from long-range dipolar coupling to sequential ¹⁴N spins in a peptide chain (¹⁴N_i–¹³C^α_i–¹⁴N_i+1 and ¹⁴N_i+1–¹³C′_i–¹⁴N_i three-spin systems) as well as residual quadrupolar–dipolar coupling cross-terms is analyzed numerically.     

Measurement of Relaxation Rates of N and H Backbone Protons in Proteins with Tailored Initial Conditions

Several methods are presented for the selective determination of spin–lattice and spin–spin relaxation rates of backbone protons in labeled proteins. The relaxation rates of amide protons in ¹⁵N labeled proteins can be measured by using two-way selective cross-polarization (SCP). The measurement of H^α relaxation rates can be achieved by combining this method with homonuclear Hartmann–Hahn transfer using doubly selective irradiation. Various schemes for selective or nonselective inversion of the longitudinal proton magnetization lead to different initial recovery rates. The methods have been applied to lysine K6 in ¹⁵N-labeled human ubiquitin and to leucine L5 in ¹⁵N- and ¹³C-labeled octapeptide YG*G*F*LRRI (GFL) in which the marked residues are ¹⁵N- and ¹³C-labeled.     

3D NMR Experiments for MeasuringN Relaxation Data of Large Proteins: Application to the 44 kDa Ectodomain of SIV gp41

A suite of 3D NMR experiments for measuring¹⁵N–{¹H} NOE,¹⁵NT₁, and¹⁵NT_1ρvalues in large proteins, uniformly labeled with¹⁵N and¹³C, is presented. These experiments are designed for proteins that exhibit extensive spectral overlap in the 2D¹H–¹⁵N HSQC spectrum. The pulse sequences are readily applicable to perdeuterated samples, which increases the spectral resolution and signal-to-noise ratio, thereby permitting the characterization of protein dynamics to be extended to larger protein systems. Application of the pulse sequences is demonstrated on a perdeuterated¹³C/¹⁵N-labeled sample of the 44 kDa ectodomain of SIV gp41.     

High-resolution solid-state NMR of anisotropically mobile molecules under very low-power H decoupling and moderate magic-angle spinning

We show that for observing high-resolution heteronuclear NMR spectra of anisotropically mobile systems with order parameters less than 0.25, moderate magic-angle spinning (MAS) rates of 11 kHz combined with ¹H decoupling at 1–2 kHz are sufficient. Broadband decoupling at this low ¹H nutation frequency is achieved by composite pulse sequences such as WALTZ-16. We demonstrate this moderate MAS low-power decoupling technique on hydrated POPC lipid membranes, and show that 1 kHz ¹H decoupling yields spectra with the same resolution and sensitivity as spectra measured under 50 kHz ¹H decoupling when the same acquisition times (50 ms) are used, but the low-power decoupled spectra give higher resolution and sensitivity when longer acquisition times (>150 ms) are used, which are not possible with high-power decoupling. The limits of validity of this approach are explored for a range of spinning rates and molecular mobilities using more rigid membrane systems such as POPC/cholesterol mixed bilayers. Finally, we show ¹⁵N and ¹³C spectra of a uniaxially diffusing membrane peptide assembly, the influenza A M2 transmembrane domain, under 11 kHz MAS and 2 kHz ¹H decoupling. The peptide ¹⁵N and ¹³C intensities at low-power decoupling are 70–80% of the high-power decoupled intensities. Therefore, it is possible to study anisotropically mobile lipids and membrane peptides using liquid-state NMR equipment, relatively large rotors, and moderate MAS frequencies.     

The 2D {P} Spin-Echo-Difference Constant-Time [C, H]-HMQC Experiment for Simultaneous Determination of JH3′P and JC4′P in C-Labeled Nucleic Acids and Their Protein Complexes

A two-dimensional {³¹P} spin-echo-difference constant-time [¹³C, ¹H]-HMQC experiment (2D {³¹P}-sedct-[¹³C, ¹H]-HMQC) is introduced for measurements of ³J_C4′P and ³J_H3′P scalar couplings in large ¹³C-labeled nucleic acids and in DNA–protein complexes. This experiment makes use of the fact that ¹H–¹³C multiple-quantum coherences in macromolecules relax more slowly than the corresponding ¹³C single-quantum coherences. ³J_C4′P and ³J_H3′P are related via Karplus-type functions with the phosphodiester torsion angles β and ε, respectively, and their experimental assessment therefore contributes to further improved quality of NMR solution structures. Data are presented for a uniformly ¹³C, ¹⁵N-labeled 14-base-pair DNA duplex, both free in solution and in a 17-kDa protein–DNA complex.     

Optimal control based NCO and NCA experiments for spectral assignment in biological solid-state NMR spectroscopy

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of (13)C,(15)N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically (15)N to (13)C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly (13)C,(15)N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical (15)N-(13)C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D (15)N-->(13)C coherence transfer experiments, as well as 2D and 3D (15)N,(13)C and (15)N,(13)C,(13)C chemical shift correlation experiments on uniformly (13)C,(15)N-labeled ubiquitin.