Separation of anisotropy and exchange broadening using (15)N CSA-(15)N-(1)H dipole-dipole relaxation cross-correlation experiments

Based on the measurement of cross-correlation rates between (15)N CSA and (15)N-(1)H dipole-dipole relaxation we propose a procedure for separating exchange contributions to transverse relaxation rates (R(2) = 1/T(2)) from effects caused by anisotropic rotational diffusion of the protein molecule. This approach determines the influence of anisotropy and chemical exchange processes independently and therefore circumvents difficulties associated with the currently standard use of T(1)/T(2) ratios to determine the rotational diffusion tensor. We find from computer simulations that, in the presence of even small amounts of internal flexibility, fitting T(1)/T(2) ratios tends to underestimate the anisotropy of overall tumbling. An additional problem exists when the N-H bond vector directions are not distributed homogeneously over the surface of a unit sphere, such as in helix bundles or beta-sheets. Such a case was found in segment 4 of the gelation factor (ABP 120), an F-actin cross-linking protein, in which the diffusion tensor cannot be calculated from T(1)/T(2) ratios. The (15)N CSA tensor of the residues for this beta-sheet protein was found to vary even within secondary structure elements. The use of a common value for the whole protein molecule therefore might be an oversimplification. Using our approach it is immediately apparent that no exchange broadening exists for segment 4 although strongly reduced T(2) relaxation times for several residues could be mistaken as indications for exchange processes.     

New (15)N NMR exchange experiments for the unambiguous assignment of (1)H(N)/(15)N resonances of proteins in complexes in slow chemical exchange with free form

The potentialities of a 2D proton-detected heteronuclear exchange experiment to assign the nitrogen and amide proton resonances in a uniformly (15)N-enriched macromolecule involved in a complex, starting from the free form assignments, are demonstrated on a protein-DNA complex. This 2D experiment is further extended to a 3D experiment in the case of severe superpositions.     

Intermolecular (1)H[(19)F] NOEs in studies of fluoroalcohol-induced conformations of peptides and proteins

Mixtures of fluorinated alcohols and water can selectively stabilize certain secondary structures of peptides and proteins. Such mixtures may also be of use in solubilizing hydrophobic or membrane-bound proteins. We show that intermolecular dipolar interactions between the fluorine nuclei of such solvents and the protons of a dissolved protein lead to readily detected (1)H[(19)F] nuclear Overhauser effects. These NOEs can potentially provide information about solvent exposure of particular groups as well as indicate the formation of long-lived fluoroalcohol-solute complexes. Results obtained with HEW lysozyme in solutions containing trifluoroethanol illustrate these possibilities.     

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.     

Characterization of 15N chemical shift tensors via 15N-13C REDOR and 1N-1H dipolar-shift CPMAS NMR spectroscopy

As part of our studies on the characterization of 15N chemical shift anisotropy (CSA) via magic angle spinning (MAS) NMR spectroscopy, we have investigated via numerical simulations the sensitivity of two different REDOR experimental protocols to the angles defining the orientation of the 15N-13C' bond vector in the principal axis system of the 15N CSA tensor of the amide nitrogen in a peptide bond. Additionally, employing polycrystalline samples of 15N and 13C', 15N-labeled acetanilide, we have obtained, in a first study of this type, the orientation of the 15N CSA tensor in the molecular frame by orienting the tensor with respect to the 15N-3C' and 15N-1H dipolar vectors via 15N-13C' REDOR and 15N-1H dipolar-shift MAS experiments, respectively.     

Sensitivity-enhanced E.COSY-type HSQC experiments for accurate measurements of one-bond 15N-1H(N) and 15N-13C' and two-bond 13C'-1H(N) residual dipolar couplings in proteins

Novel E.COSY-type HSQC experiments are presented for the accurate measurement of one-bond 15N-1H(N) and 15N-13C(') and two-bond 13C(')-1H(N) residual dipolar couplings in proteins. Compared with existing experiments, the (delta,J)-E.COSY experiments described here are composed of fewer pulses and the resulting spectra exhibit 1.4 times the sensitivity of coupled HSQC spectra. Since residual dipolar couplings play increasingly important roles in structural NMR, the proposed methods should find wide spread application for structure determination of proteins and other biological macromolecules.     

Uptake of [(15)N] Ammonium and [(15)N]Nitrate in a 140-Year-Old Spruce Stand (Picea abies) in the Fichtelgebirge (NE Bavaria)

Abstract In April 1994 a (15)N tracer pulse study was started in a 140-year-old spruce stand (Picea abies [L.] Karst.) located at the Fichtelgebirge (NE Bavaria). Highly enriched (98%) [(15)N]ammonium and [(15)N]nitrate were applied simulating wet deposition. For two growing seasons the pathways and dynamics of the tracer were followed in all compartments of spruce (needles and twigs of all age classes, stem wood and bark, roots) and understorey vegetation and in soils of the organic (L/Of and Oh) and mineral horizons (A(0-5) and A(5-10)). By variations of the application time on different plots within the growing season (spring, summer and autumn) a seasonal effect of labelling on uptake and distribution patterns was tested. First results of this tracer study indicate that young and old spruce stands do not differ basically in pattern of uptake and distribution of mineral nitrogen. There are indications that spruce uses preferentially ammonium versus nitrate and that the ratio of ammonium/nitrate which is being consumed depends on the ammonium/nitrate ratio in the soil solution. The uptake rates decrease within the growing season.     

The Fate of [(15)N]Ammonium and [(15)N]Nitrate in the Soil of a 140-Year-Old Spruce Stand (Picea Abies) in the Fichtelgebirge (NE-Bavaria)

Abstract A (15)N tracer-experiment was carried out in a 140-year-old spruce stand (Picea abies (L.) Karst.) in the Fichtelgebirge (NE-Bavaria, Germany). Highly enriched (98 at%) [(15)N]ammonium and [(15)N]nitrate were applied as tracers by simulation of a deposition of 41.3 mol N ha(-1) with 11 water m(-2). To examine seasonal variations of uptake by spruce and understorey vegetation, different plots were labelled in spring, summer and autumn 1994. One aim of the present study was to perfect a method of preparation of soil extracts for isotope ratio mass spectrometry (IRMS) measurements. Ammonium and nitrate from soil extracts were prepared for IRMS measurements by steam distillation and subsequent freeze drying. Additionally, tracer distribution and transformations in the soil nitrogen pools were examined. Ammonium, nitrate and total nitrogen were examined in the organic layer and the upper 10 cm of the mineral soil during 3 months after the first tracer application in spring 1994. In July 1994, three months after tracer application, 40% of the [(15)N]ammonium label and 29% of the [(15)N]nitrate label, respectively, were recovered in the total N pool of the investigated soil horizons. In the organic layer the L/Of horizon retained most of the recovered tracers. Nitrification, immobilisation and mineralisation occurred even under the conditions of high soil acidity at the study site.     

On the measurement of 15N-{1H} nuclear Overhauser effects

Accurate quantification of the 15N-{1H} steady-state NOE is central to current methods for the elucidation of protein backbone dynamics on the fast, sub-nanosecond time scale. This experiment is highly susceptible to systematic errors arising from multiple sources. The nature of these errors and their effects on the determined NOE ratio is evaluated by a detailed analysis of the spin dynamics during the pair of experiments used to measure this ratio and possible improvements suggested. The experiment that includes 1H irradiation, is analyzed in the framework of Average Liouvillian Theory and a modified saturation scheme that generates a stable steady-state and eliminates the need to completely saturate 1H nuclei is presented. The largest source of error, however, in 1H-dilute systems at ultra-high fields is found to be an overestimation of the steady-state NOE value as a consequence of the incomplete equilibration of the magnetization in the so-called "reference experiment". The use of very long relaxation delays is usually an effective, but time consuming, solution. Here, we introduce an alternative reference experiment, designed for larger, deuterated systems, that uses the fastest relaxing component of the longitudinal magnetization as a closer approximation to the equilibrium state for shorter relaxation delays. The utility of the modified approach is illustrated through simulations on realistic spin systems over a wide range of time scales and experimentally verified using a perdeuterated sample of human ubiquitin.     

Determination of internucleotide (h)J(HN) couplings by the modified 2D J(NN)-correlated [(15)N, (1)H] TROSY

Recent developments in the direct observation of J couplings across hydrogen bonds in proteins and nucleic acids provide additional information for structure and function studies of these molecules by NMR spectroscopy. A J(NN)-correlated [(15)N, (1)H] TROSY experiment proposed by Pervushin et al. (Proc. Natl. Acad. Sci. USA 95, 14147-14151, 1998) can be applied to measure (h)J(HN) in smaller nucleic acids in an E.COSY manner. However, it cannot be effectively applied to large nucleic acids, such as tRNA(Trp), since one of the peaks corresponding to a fast relaxing component will be too weak to be observed in the spectra of large molecules. In this Communication, we proposed a modified J(NN)-correlated [(15)N, (1)H] TROSY experiment which enables direct measurement of (h)J(HN) in large nucleic acids.     

Sensitivity-enhanced double-TROSY experiment for simultaneous measurement of one-bond 15N-1H, 15N-13C' and two-bond 1H-13C' couplings

A recently published experiment for the measurement of 1JHN, 1JNC', and 2JHC' coupling constants [J. Am. Chem. Soc. 125 (2003) 11504] was modified to yield a double-TROSY experiment which selects 1 of the 16 multiplet components from a 15N-HSQC spectrum recorded of a uniformly 15N/13C-labelled protein. Subspectra containing any 1 of the 16 multiplet components can be generated allowing accurate coupling constant measurements. The experiment is sensitivity enhanced, turning all magnetization components precessing during the evolution time into observable magnetization during the detection time. The experiment is discussed with regard to the previously published alpha/beta-filtered HN(alpha/beta-NC'-J) experiment [J. Magn. Reson. 140 (1999), 32] which measures the same coupling constants.     

Improved measurement of (3)J(H(alpha)(i),N(i+1)) coupling constants in H(2)O dissolved proteins.

A modification to the recently proposed alpha/beta-HN(CO)CA-J TROSY pulse sequence (P. Permi et al., J. Magn. Reson. 146, 255-259 (2000)) makes it possible to determine (3)J(H(alpha)(i), N(i+1)) coupling constants from a single E.COSY-type cross-peak pattern rather than from two (1)H(alpha) spin-state-edited subspectra. Advantages are increased (15)N resolution, critical to extracting accurate (1)H(alpha)-(15)N coupling constants, and minimized differential relaxation due to nested (13)C(alpha) and (15)N evolution periods. Application of the improved pulse sequence to Desulfovibrio vulgaris flavodoxin results in (3)J(H(alpha)(i), N(i+1)) values being systematically larger than those obtained with the original scheme. Parametrization of the coupling dependence on the protein backbone torsion angle psi yields the Karplus relation (3)J(H(alpha)(i), N(i+1))=-1.00 cos(2)(psi-120 degrees )+0.65 cos(psi-120 degrees )-0.15 Hz, with a residual root-mean-square difference of 0.13 Hz between measured and back-calculated coupling constants. The curve compares with data derived from ubiquitin (A. C. Wang and A. Bax, J. Am. Chem. Soc. 117, 1810-1813 (1995)), although spanning a slightly larger range of J values in flavodoxin. The orientation of the Ala39/Ser40 peptide link, forming a type-II beta-turn in flavodoxin, is twisted against X-ray-derived torsions by approximately 10 degrees in the NMR structure as evident from the analysis of straight phi- and psi-related (3)J coupling constants. The remaining deviation of some experimental values from the prediction is likely to be due to strong hydrogen bonding, substituent effects, or the additional dependence on the adjacent torsions straight phi.     

Three-dimensional protein NMR TROSY-type (15)N-resolved (1)H(N)-(1)H(N) NOESY spectra with diagonal peak suppression

An earlier two-dimensional NOESY experiment with diagonal peak suppression in the (1)H(N)-(1)H(N) region is extended to three dimensions by including (15)N evolution while maintaining the TROSY approach throughout. The technique suppresses all anti-TROSY resonances by appropriate pulse sequence elements and for large molecules at high fields possible semi- and anti-TROSY artifacts are further suppressed by virtue of much shorter transverse relaxation times for these components. The new technique is demonstrated using an (15)N-labeled protein sample, RAP 17-97 (N-terminal domain of alpha2-macroglobulin Receptor Associated Protein), in H(2)O at 500 MHz.     

2,3-二羟基萘钼多酸有机衍生物的合成及1H NMR和IR谱学研究

本文对二种新合成的2，3－二羟基萘二钼和四钼多酸有机衍生物[n-Bu)4N]2[Mo2O5(OC10H6O)2](Ⅰ）和[n-Bu)4N]2[Mo4O10(OC10H6O)2(OCH3)2](Ⅱ)进行了红外光谱与核磁共振波谱研究，发现[Mo2O5]^2 中钼氧多桥键的红外振动频率较[Mo4O10(OCH3)2]^2 中钼氧多桥键的红外振动频率红移，而在配合物Ⅱ中2，3－二羟基中芳环的^1H化学位移较配合物Ⅰ中向低场移动。同时还发现含二钼配位中心[Mo2O5]^2 的[Mo2O5(OC10H6O)2]^2-与含四钼配位中心[Mo4O10(OCH3)2]^2 的[Mo4O10(OC10H6O)2(OCH3)2]^2-生成条件的差异仅仅只在反应体系的pH值的微小变化，说明钼多酸有机衍生物阴离子是对体系酸碱度极为敏感的物质。     

Semi-constant-time HMSQC (SCT-HMSQC-HA) for the measurement of (3)J(H(N))(H(alpha)) couplings in (15)N-labeled proteins

A simple method for accurately measuring (3)J(H(N))(H(alpha)) coupling constants in (15)N-labeled proteins is described. This semi-constant-time HMSQC-HA experiment combines the rapidity and convenience of the recently introduced CT-HMQC-HA scheme (Postingl and Otting, J. Biomol. NMR 12, 319-324 (1998)) with the high resolution and robustness of the HSQC experiment. The proposed method is demonstrated for the 76-residue human ubiquitin and Saccharopolyspora erythraea calerythrin (176 residues). Our results imply that the SCT-HMSQC-HA experiment is suitable also for proteins with less favorable NMR properties due to its good resolution and sensitivity.     

NMR proton relaxation and chemical exchange in the system H16 2O/H17 2O-[2H6]dimethylsulphoxide

NMR proton relaxation rates of normal and ¹⁷O enriched water in a mixture of 68 mol% water and 32 mol% [²H₆]dimethylsulphoxide were measured for temperatures between 298 K and 183 K. In the range between 240 K and 204 K the limit of fast proton-proton exchange between H¹⁶ ₂O and H¹⁷ ₂O is not obeyed, and relaxation curves deviate from mono-exponential behaviour. By fitting the relaxation curves to a model of NMR two-phase relaxation the proton-proton exchange rate within the aqueous component could be obtained. With decreasing temperature, proton-proton exchange slows down and a residence time of about 125 ms at 215 K is found, but it becomes faster again for still lower temperatures. From the phase-averaged relaxation rates of water in the ¹⁷O enriched mixtures, the ¹⁷O induced proton relaxation rate was derived as a function of temperature. This yields the rotational correlation times of the water molecule in the mixture and the dipolar spin-lattice coupling parameter. The latter is considerably lower than the one predicted from the geometry of water.     

Accurate measurement of H(N)-H(alpha) residual dipolar couplings in proteins.

A method for accurately measuring H(N)-H(alpha) residual dipolar couplings is described. Using this technique, both the sign and magnitude of the coupling can be determined easily. Residual dipolar coupling between H(N)(i)-H(alpha)(i) and H(N)(i)-H(alpha)(i-1) were measured for the FK506 binding protein complexed to FK506. The experimental values were in excellent agreement with predictions based on an X-ray crystal structure of the protein/ligand complex, suggesting that these residual dipolar couplings will provide accurate structural constraints for the refinement of protein structures determined by NMR.     

A15N-1H dipolar CSA solid-state NMR study of polymorphous polyglycine (-CO-CD2-15NH-)n

The solid-state¹H MAS (magic-angle spinning),²H static,¹⁵N CP (cross polarization)-MAS and¹⁵N-¹H dipolar CSA (chemical shielding anisotropy) NMR (nuclear magnetic resonance) spectra of two different modifications of C_α-deuterated^l5N-polyglycine, namely PG I and PG II (-CO-CD₂-^l5NH-)_n are measured. The data from these spectra are compared to previous NMR, infrared, Raman and inelastic neutron scattering work. The deuteration of C_α eliminates the largest intramolecular¹H-¹H dipolar coupling. The effect of the remaining (N)H-(N)H interaction (～5 kHz) is not negligible compared to the¹⁵N-¹H coupling (about 10 kHz). Its effect on the dipolar CSA spectra, described as a two-spin system, is analyzed analytically and numerically and it is shown that those parts of the powder spectrum, which correspond to orientations with a strong dipolar¹⁵N-¹H interaction, can be described as an effective two-spin system, permitting the measurement of the strength of the¹⁵N-¹H dipolar interaction and the orientation of the dipolar vector with respect to the¹⁵N CSA frame. While in the PG II system the¹⁵N CSA tensor is collinear with the amide plane, in the PG I system the CSA tensor is tilted ca. 16° with respect to the (δ₁₁δ₂₂) CSA plane.     

Kinetic C(2)-H exchange study ofcis-[(en)2Co(N-MeIm)OPO3H2]2+ by NMR: Evidence for the acidity of C(2)-H proton

The complexcis-[(en)₂Co(N-MeIm)OPO₃H₂]Br₂ (en=ethylenediamine, Me=methyl, Im=imidazole) has been synthesized from [(en)₂Co(N-MeIm)Br]Br₂, anhydrous AgCIO₄, and H₃PO₄ (0.85%), and characterized by¹H and¹³C nuclear magnetic resonance spectroscopy. C_(2)-H exchange in this complex was studied over the pD range from 9.40 to 12.80 at 60 °C, wherecis-[(en)₂Co(N-MeIm)OPO₃D]⁺ andcis-[(en)₂Co(N-MeIm)OPO₃]⁰ are the kinetically important species. The kinetic data may be interpreted in terms of an intramolecular interaction of the −OPO₃²⁻ group and H-2 in the coordinated N-MeIm.