Determination of three-bond scalar coupling between 13C' and 1H(alpha) in proteins using an iHN(CA),CO(alpha/beta-J-COHA) experiment

Triple-resonance NMR experiments for measuring three-bond scalar coupling constant between 13C' (i-1) and 1H(alpha)(i) spins, defining the dihedral angle phi, are presented. The novel experiments enable the measurement of 3JC'H(alpha)) from simple two (or three)-dimensional 13C', (15N/13C(alpha)), 1H(N) correlation spectra with minimal resonance overlap, thanks to solely intraresidual coherence transfer pathway and spin-state-selection. The 3J(C'H(alpha)) values measured in human ubiquitin using the proposed intraresidual iHN(CA),CO(alpha/beta-J-COHA) TROSY method were compared with those determined previously utilizing the HCAN[C'] experiment.     

3hJ coupling between C(alpha) and H(N) across hydrogen bonds in proteins

J couplings between (13)C(alpha) and (1)H(N) across hydrogen bonds in proteins are reported for the first time, and a two- or three-dimensional NMR technique for their measurement is presented. The technique exploits the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms, for sensitivity enhancement. The 2D or 3D spectra exhibit E.COSY patterns where the splittings in the (13)CO and (1)H(N) dimensions are (1)J((13)C(alpha), (13)CO) and the desired (3h)J((13)C(alpha), (1)H(N)), respectively. A demonstration of the new method is shown for the (15)N,(13)C-labeled protein chymotrypsin inhibitor 2 where 17 (3h)J((13)C(alpha), (1)H(N)) coupling constants ranging from 0 to 1.4 Hz where identified and all of positive sign.     

J Coupling between C and H across Hydrogen Bonds in Proteins

Two new two- or three-dimensional NMR methods for measuring ^3hJ_C′N and ^2hJ_C′H coupling constants across hydrogen bonds in proteins are presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D or 3D spectra into two separate subspectra corresponding to the two possible spin states of the ¹H^N spin during evolution of ¹³CO coherences. This allows ^2hJ_C′H to be measured in an E.COSY-type way while ^3hJ_C′N can be measured in the so-called quantitative way provided a reference spectrum is also recorded. A demonstration of the new methods is shown for the ¹⁵N,¹³C-labeled protein chymotrypsin inhibitor 2.     

Methods for the measurement of (1)J(NCalpha) and (2)J(NCalpha) from a simplified 2D (13)C(alpha)-coupled (15)N SE-HSQC spectrum

Two methods for the measurement of (2)J(NCalpha) and (1)J(NCalpha) in (15)N/(13)C-labeled small and medium-size proteins are described. The current approach is based on simplified (13)C(alpha)-coupled (15)N HSQC spectra, where the two (2)J(NCalpha) doublets are separated into two subspectra corresponding to the alpha and beta spin states of the residue's own alpha carbon. The displacement of the two (2)J(NCalpha) doublets between the two subspectra provides an accurate value for (1)J(NCalpha). The alpha/beta filtration is achieved by taking the sum and difference of the recorded complementary in-phase and antiphase J-coupled spectra. J-multiplication is utilized in one of the proposed methods. In this method, an additional coupling evolution period, which is incremented in concert with t(1), is included in the pulse sequence making it possible to scale the peak-to-peak separation.     

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.     

Determination of backbone angle psi in proteins using a TROSY-based alpha/beta-HN(CO)CA-J experiment

Transverse relaxation-optimized NMR experiment (TROSY) for the measurement of three-bond scalar coupling constant between (1)H(alpha)(i-1) and (15)N(i) defining the dihedral angle psi is described. The triple-spin-state-selective experiment allows measurement of (3)J(H(alpha)N) from (13)C(alpha), (15)N, and (1)H(N) correlation spectra H(2)O with minimum resonance overlap. Transverse relaxation of (13)C(alpha) spin is minimized by using spin-state-selective filtering and by acquiring a signal longer in (15)N-dimension in a manner of semi-constant-time TROSY evolution. The (3)J(H(alpha))(N) values obtained with the proposed alpha/beta-HN(CO)CA-J TROSY scheme are in good agreement with the values measured earlier from ubiquitin in D(2)O using the HCACO[N] experiment.     

Cross-correlations between low-γ nuclei in solids via a common dipolar bath

Correlation of chemical shifts of low-γ nuclei (such as 15N) is an important method for assignment of resonances in uniformly-labeled biological solids. Under static experimental conditions, an efficient mixing of low-γ nuclear spin magnetization can be achieved by a thermal contact to the common reservoir of dipole-dipole interactions in order to create 15N-15N, 13C-13C, or 15N-13C cross-peaks in a 2D correlation spectrum. A thermodynamic approach can be used to understand the mechanism of magnetization mixing in various 2D correlation pulse sequences. This mechanism is suppressed under magic-angle spinning, when mixing via direct cross-polarization with protons becomes more efficient. Experimental results are presented for single-crystalline and powder samples of 15N-labeled N-acetyl-L-15N-valyl-L-15N-leucine (NAVL). In addition to the thermodynamic analysis of mixing pulse sequences, two different new mixing sequences utilizing adiabatic pulses are also experimentally demonstrated.     

Efficient measurement of (3)J(N,Cgamma) and (3)J(C',Cgamma) coupling constants of aromatic residues in (13)C, (15)N-labeled proteins

An NMR pulse sequence is proposed for the simultaneous determination of side chain chi1 torsion-angle related (3)J(N,Cgamma) and (3)J(C', Cgamma) couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated (15)N and (1)H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly (13)C/(15)N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.     

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.     

Simultaneous measurement of protein one-bond residual dipolar couplings without increased resonance overlap

A NMR strategy designed to measure simultaneously and without increased resonance overlap scalar and dipolar couplings (RDCs) in (13)C-, (15)N-labeled proteins is presented. Contrary to common schemes for simultaneous measurement of RDCs, a single reference experiment is used for the extraction of more than one type of coupling, thereby reducing the required measurement time. This is accomplished by a common reference spectrum followed by a series of interleaved experiments, in which a particular coupling dependent parameter is varied according to the quantitative J-correlation method or using accordion spectroscopy. To illustrate this idea, we have modified the 3D TROSY-HNCO and the 3D CBCA(CO)NH experiment allowing efficient measurement of one-bond (1)D(NH), (1)D(C'N), (1)D(CalphaHalpha), (1)D(CbetaHbeta), and (1)D(CalphaC') couplings in small to medium sized proteins. In addition, the experiments are expected to be useful for largely unfolded proteins, which show strong resonance overlap but have very favorable relaxation properties. Measurement of RDCs is demonstrated on uniformly (15)N-(13)C-labeled ubiquitin and on the sensory domain of the membraneous two-component fumarate sensor DcuS of Escherichia coli (17 kDa). DcuS was found to be unstable and to precipitate in one to two weeks. RDCs obtained from these experiments are in good agreement with the 1.8A X-ray structure of ubiquitin.     

Specification and visualization of anisotropic interaction tensors in polypeptides and numerical simulations in biological solid-state NMR

Software facilitating numerical simulation of solid-state NMR experiments on polypeptides is presented. The Tcl-controlled SIMMOL program reads in atomic coordinates in the PDB format from which it generates typical or user-defined parameters for the chemical shift, J coupling, quadrupolar coupling, and dipolar coupling tensors. The output is a spin system file for numerical simulations, e.g., using SIMPSON (Bak, Rasmussen, and Nielsen, J. Magn. Reson. 147, 296 (2000)), as well as a 3D visualization of the molecular structure, or selected parts of this, with user-controlled representation of relevant tensors, bonds, atoms, peptide planes, and coordinate systems. The combination of SIMPSON and SIMMOL allows straightforward simulation of the response of advanced solid-state NMR experiments on typical nuclear spin interactions present in polypeptides. Thus, SIMMOL may be considered a "sample changer" to the SIMPSON "computer spectrometer" and proves to be very useful for the design and optimization of pulse sequences for application on uniformly or extensively isotope-labeled peptides where multiple-spin interactions need to be considered. These aspects are demonstrated by optimization and simulation of novel DCP and C7 based 2D N(CO)CA, N(CA)CB, and N(CA)CX MAS correlation experiments for multiple-spin clusters in ubiquitin and by simulation of PISA wheels from PISEMA spectra of uniaxially oriented bacteriorhodopsin and rhodopsin under conditions of finite RF pulses and multiple spin interactions.     

piN and pipiN couplings of the Delta(1232) and its chiral partners

We investigate the interactions and chiral properties of the four spin-3/2 baryons N(-)(D13), N+(P13), Delta+(P33), and Delta(-)(D33) together with the nucleon. We construct the SU(2)R x SU(2)L invariant interactions between the spin-1/2 and spin-3/2 baryons with the aid of a new, specially developed spin and isospin projection technique for these baryon fields, where the chiral invariant interactions contain one- and two-pion couplings. We obtain simple relations for the coupling constants of the one- and two-pion spin-1/2-3/2 transitions terms. The relation for the one-pion interactions reasonably agrees with the experiments, which suggests that these spin-3/2 baryons are chiral partners.     

13C/14N heteronuclear multiple-quantum correlation with rotary resonance and REDOR dipolar recoupling

A two-dimensional (13)C/(14)N heteronuclear multiple quantum correlation (HMQC) experiment using dipolar recoupling under magic-angle spinning (MAS) is described. The experiment is an extension of the recent indirect (13)C detection scheme for measuring (14)N quadrupolar coupling under MAS. The recoupling allows the direct use of the much larger dipolar interaction instead of the small J and residual dipolar couplings for establishing (13)C/(14)N correlations. Two recoupling methods are incorporated into the HMQC sequence, both applying rf only to the observed (13)C spin. The first one uses the REDOR sequence with two pi-pulses per rotor cycle. The second one uses a cw rf field matching the spinning frequency, known as rotary resonance. The effects of CSA, T(2)(') signal loss, MAS frequency and stability and t(1)-noise are compared and discussed.     

Pulse sequences for measurement of one-bond (15)N-(1)H coupling constants in the protein backbone.

A set of three improved two-dimensional (2D) NMR methods for measuring one-bond (15)N-(1)H coupling constants in the protein backbone is presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D spectra into two separate subspectra corresponding to the two possible spin states of the coupling partner. Cross talk between the two subspectra is a second order effect in the difference between the actual coupling constants and the one used in setting the pertinent delays of the pulse sequences. This relatively high degree of editing accuracy makes the methods useful for applications to molecules subjected to weak alignment where the one-bond coupling constants are linear combinations of a scalar J and a residual dipolar contribution containing important structural information. A demonstration of the new methods is shown for the (15)N-labeled protein chymotrypsin inhibitor 2 in a lipid bicelle mixture.     

A frustrated three-dimensional antiferromagnet: stacked J1-J2 layers

We study a frustrated 3D antiferromagnet of stacked J(1)-J(2) layers. The intermediate 'quantum spin liquid' phase, present in the 2D case, narrows with increasing interlayer coupling and vanishes at a triple point. Beyond this, there is a direct first-order transition from Néel to columnar order. Possible applications to real materials are discussed.     

Hysteresis in the random-field Ising model and bootstrap percolation

We study hysteresis in the random-field Ising model with an asymmetric distribution of quenched fields, in the limit of low disorder in two and three dimensions. We relate the spin flip process to bootstrap percolation, and show that the characteristic length for self-averaging L small star, filled increases as exp[exp(J/Delta)] in 2D, and as exp(exp[exp(J/Delta)]) in 3D, for disorder strength Delta much less than the exchange coupling J. For system size 1infinity for both square and cubic lattices. For lattices with coordination number 3, the limiting magnetization shows no jump, and h(coer) tends to J.     

2D(13)C-(13)C MAS NMR correlation spectroscopy with mixing by true (1)H spin diffusion reveals long-range intermolecular distance restraints in ultra high magnetic field

An improved 2D (13)C-(13)C CP(3) MAS NMR correlation experiment with mixing by true (1)H spin diffusion is presented. With CP(3), correlations can be detected over a much longer range than with direct (1)H-(13)C or (13)C-(13)C dipolar recoupling. The experiment employs a (1)H spin diffusion mixing period tau(m) sandwiched between two cross-polarization periods. An optimized CP(3) sequence for measuring polarization transfer on a length scale between 0.3 and 1.0 nm using short mixing times of 0.1 ms < tau(m) < 1 ms is presented. For such a short tau(m), cross talk from residual transverse magnetization of the donating nuclear species after a CP can be suppressed by extended phase cycling. The utility of the experiment for genuine structure determination is demonstrated using a self-aggregated Chl a/H(2)O sample. The number of intramolecular cross-peaks increases for longer mixing times and this obscures the intermolecular transfer events. Hence, the experiment will be useful for short mixing times only. For a short tau(m) = 0.1 ms, intermolecular correlations are detected between the ends of phytyl tails and ring carbons of neighboring Chl a molecules in the aggregate. In this way the model for the structure, with stacks of Chl a that are arranged back to back with interdigitating phytyl chains stretched between two bilayers, is validated.     

Sensitivity-enhanced MQ-HCN-CCH-TOCSY and MQ-HCN-CCH-COSY pulse schemes for (13)C/(15)N labeled RNA oligonucleotides

Sensitivity enhanced multiple-quantum 3D HCN-CCH-TOCSY and HCN-CCH-COSY experiments are presented for the ribose resonance assignment of (13)C/(15)N-labeled RNA sample. The experiments make use of the chemical shift dispersion of N1/N9 of pyrimidine/purine to distinguish the ribose spin systems. They provide a complementary approach for the assignment of ribose resonance to the currently used HCCH-COSY and HCCH-TOCSY type experiments in which either (13)C or (1)H is utilized to separate the different ribose spin systems. The pulse schemes have been demonstrated on a 23-mer (13)C/(15)N-labeled RNA aptamer complexed with neomycin and tested on a 32-mer RNA complexed with a 23-residue peptide.     

Heteronuclear polarization transfer by symmetry-based recoupling sequences in solid-state NMR

We demonstrate a new set of methods for transferring spin polarization between different nuclear isotopes in magic-angle-spinning solid-state NMR. The technique employs symmetry-based recoupling sequences on one irradiation channel and a simple sequence of between one and three strong radiofrequency pulses on the second channel. A phase shift of the recoupling sequences is applied at the same time as a pi/2 pulse on the second channel. The trajectory of the transferred polarization may be used to estimate heteronuclear distances. The method is particularly attractive for nuclei with low gyromagnetic ratios or for those experiencing strong anisotropic spin interactions, where conventional Hartmann-Hahn cross-polarization is difficult to apply. We demonstrate the method on 1H-13C, 1H-15N and 19F-109Ag systems.     

Observing in-phase single-quantum 15N multiplets for NH2/NH3+ groups with two-dimensional heteronuclear correlation spectroscopy

Two-dimensional (2D) F1-(1)H-coupled HSQC experiments provide 3:1:1:3 and 1:0:1 multiplets for AX(3) and AX(2) spin systems, respectively. These multiplets occur because, in addition to the 2S(y)H(z)(a)-->2S(y)H(z)(a) process, the coherence transfers such as 2S(y)H(z)(a)-->2S(y)H(z)(b) occurring in t(1) period provide detectable magnetization during the t(2) period. Here, we present a 2D F1-(1)H-coupled (1)H-(15)N heteronuclear correlation experiment that provides a 1:3:3:1 quartet for AX(3) spin system and a 1:2:1 triplet for AX(2). The experiment is a derivative of 2D HISQC experiment [J. Iwahara, Y.S. Jung, G.M. Clore, Heteronuclear NMR spectroscopy for lysine NH(3) groups in proteins: unique effect of water exchange on (15)N transverse relaxation. J. Am. Chem. Soc. 129 (2007) 2971-2980] and contains a scheme that kills anti-phase single-quantum terms generated in the t(1) period. The purge scheme is essential to observe in-phase single-quantum multiplets. Applications to the NH(2) and NH(3)(+) groups in proteins are demonstrated.