15N Chemical Shift Anisotropy Research Articles

Variability of the 15N Chemical Shift Anisotropy in Escherichia coli Ribonuclease H in Solution

The 15N chemical shift anisotropy, Δσ, is reported for 81 well-ordered backbone amide sites in the Escherichia coli enzyme ribonuclease H. The values of Δσ were determined from 15N relaxation rate constants measured at static magnetic field strengths of 11.7, 14.1, and 18.7 T; the data analyzed included both autorelaxation rate constants and 1H−15N dipolar/15N chemical shift anisotropy relaxation interference rate constants. For this data set, the values of Δσ are approximately Gaussian distributed with a mean of −172 ± 13 ppm. The standard deviation of the site-to-site variation of the chemical shift anisotropy is 5.5 ppm and a 95% confidence limit for this variation is 9.6 ppm. The site-to-site variation in the chemical shift anisotropy is similar to the variation observed in solid state NMR studies of specifically labeled polypeptides. Variability in the value of Δσ becomes a significant factor in the interpretation of spin relaxation rate constants at 18.7 T, but is less significant at lower field str...

An Approach to Direct Determination of Protein Dynamics from 15N NMR Relaxation at Multiple Fields, Independent of Variable 15N Chemical Shift Anisotropy and Chemical Exchange Contributions

An approach to protein dynamics analysis from 15N relaxation data is demonstrated, based on multiple-field relaxation data. This provides a direct, residue-specific determination of both the spectral density components, the 15N chemical shift anisotropy (CSA) and the conformational exchange contribution to the 15N line width. Measurements of R1, R2, and 15N{1H} NOE are used. The approach is free from any assumption about the values of the CSA or of the conformational exchange. Using this approach, the spectral densities, the values of 15N CSA, and the conformational exchange contribution to the 15N line width are directly determined from the relaxation data for human ubiquitin, collected at 360, 500, and 600 MHz. The spectral densities are analyzed in terms of the order parameter and the correlation time of local motion, using an axially symmetric overall rotational diffusion model. The residue-specific values of 15N CSA and the spectral densities obtained using this approach are in agreement with those d...

The effect of noncollinearity of 15N-1H dipolar and 15N CSA tensors and rotational anisotropy on 15N relaxation, CSA/dipolar cross correlation, and TROSY.

Current approaches to 15N relaxation in proteins assume that the 15N-1H dipolar and 15N CSA tensors are collinear. We show theoretically that, when there is significant anisotropy of molecular rotation, different orientations of the two tensors, experimentally observed in proteins, nucleic acids, and small peptides, will result in differences in site-specific correlation functions and spectral densities. The standard treatments of the rates of longitudinal and transverse relaxation of amide 15N nuclei, of the 15N CSA/15N-1H dipolar cross correlation, and of the TROSY experiment are extended to account for the effect of noncollinearity of the 15N-1H dipolar and 15N CSA (chemical shift anisotropy) tensors. This effect, proportional to the degree of anisotropy of the overall motion, (D parallel/D perpendicular - 1), is sensitive to the relative orientation of the two tensors and to the orientation of the peptide plane with respect to the diffusion coordinate frame. The effect is negligible at small degrees of anisotropy, but is predicted to become significant for D parallel/D perpendicular > or = 1.5, and at high magnetic fields. The effect of noncollinearity of 15N CSA and 15N-1H dipolar interaction is sensitive to both gross (hydrodynamic) properties and atomic-level details of protein structure. Incorporation of this effect into relaxation data analysis is likely to improve both precision and accuracy of the derived characteristics of protein dynamics, especially at high magnetic fields and for molecules with a high degree of anisotropy of the overall motion. The effect will also make TROSY efficiency dependent on local orientation in moderately anisotropic systems.

Measurement of 3hJNC' connectivities across hydrogen bonds in a 30 kDa protein.

A method is described which permits detection of 3hJNC' scalar couplings across hydrogen bonds in larger, perdeuterated proteins. The experiment is demonstrated for the uniformly 2H/13C/15N-enriched 30 kDa ribosome inactivating protein MAP30. The 3hJNC' interactions are smaller than 1 Hz, but their detection in an HNCO experiment is made possible through the use of constructive interference between the 15N chemical shift anisotropy and 1H-15N dipole-dipole relaxation mechanisms in a manner similar to that of recently proposed TROSY schemes. Sensitivity of the HNCO experiment depends strongly on the 15N transverse relaxation rate of the downfield 15N multiplet component and on the amide proton T1. In perdeuterated MAP30 at 40 degrees C, the average TROSY T2 was 169 ms at 750 MHz 1H frequency, and a wide range of longitudinal relaxation rates was observed for the amide protons.

Assessing potential bias in the determination of rotational correlation times of proteins by NMR relaxation.

The various factors that influence the reliable and efficient determination of the correlation time describing molecular reorientation of proteins by NMR relaxation methods are examined. Nuclear Overhauser effects, spin-lattice, and spin-spin relaxation parameters of 15N NMR relaxation in ubiquitin have been determined at 17.6, 14.1, 11.7 and 9.4 Tesla. This unusually broad set of relaxation parameters has allowed the examination of the influence of chemical shift anisotropy, the functional form of the model-free spectral density, and the reliability of determined spin-spin relaxation parameters on the characterization of global tumbling of the protein. Treating the 15N chemical shift anisotropy (CSA) as an adjustable parameter, a consensus value of -170 +/- 15 ppm for the breadth of the chemical shift tensor and a global isotropic correlation time of 4.1 ns are found when using the model-free spectral density to fit T1 and NOE data from all fields. The inclusion of T2 relaxation parameters in the determination of the global correlation time results in its increase to 4.6 ns. This apparent inconsistency may explain a large portion of the discrepancy often found between NMR- and fluorescence-derived tau m values for proteins. The near identity of observed T2 and T1 rho values suggests that contributions from slow motions are not the origin of the apparent inconsistency with obtained T1 and NOE data. Various considerations suggest that the origin of this apparent discrepancy may reside in a contribution to the spectral density at zero frequency that is not represented by the simple model-free formalism in addition to the usual experimental difficulties associated with the measurement of these relaxation parameters. Finally, an axially symmetric diffusion tensor for ubiquitin is obtained using exclusively T1 and NOE data. A recommendation is reached on the types and combinations of relaxation data that can be used to reliably determine tau m values. It is also noted that the reliable determination of tau m values from 15N T1 and NOE relaxation parameters will become increasingly difficult as tau m increases.

Direct Measurement of 15N Chemical Shift Anisotropy in Solution

The magnitude and orientation of the 15N chemical shift anisotropy (CSA) tensors are determined for human ubiquitin in solution from 15N relaxation data at 600, 500, and 360 MHz. The analysis uses the model-independent approach [Fushman, D.; Cowburn, D. J. Am. Chem. Soc. 1998, 120, 7109−10] based on a ratio, η/R2, of the cross correlation (η) between 15N CSA and 15N−1H dipolar interaction and of the rate (R2) of 15N transverse relaxation. Since the η/R2 ratio does not contain any direct dependence on protein dynamics, the present approach is free from assumptions about overall and local motions. The 15N CSA values fall in the range −125 to −216 ppm, with the average value of −157 ± 19 ppm; the average angle between the NH bond and the unique principal axis of the 15N CSA tensor was 15.7 ± 5.0° (range 6−26°). The results indicate the importance of residue-specific 15N CSA for accurate analysis of dynamics from relaxation data, and provide access to the CSA in solution, which may be structurally useful.

Longitudinal and Transverse 1H−15N Dipolar/15N Chemical Shift Anisotropy Relaxation Interference: Unambiguous Determination of Rotational Diffusion Tensors and Chemical Exchange Effects in Biological Macromolecules

High-resolution proton-detected heteronuclear correlation NMR spectroscopy allows the measurement of 15N spin relaxation rates at multiple sites throughout a biological macromolecule. The rate constants are determined by stochastic internal motions on time scales of picoseconds to nanoseconds, overall molecular rotational diffusion on time scales of nanoseconds, and chemical exchange rates on time scales of microseconds to milliseconds. A new method has been developed for distinguishing the contributions of chemical exchange from the contributions due to anisotropic rotational diffusion by measuring both longitudinal and transverse interference between the 1H−15N dipolar and 15N chemical shift anisotropy interactions. The spectroscopic experiment for measuring the longitudinal cross-correlation rate constant for 1H−15N dipolar/15N chemical shift anisotropy interference is based on the approach for measuring the transverse cross-correlation rate constant (Tjandra, N.; Szabo, A.; Bax, A. J. Am. Chem. Soc. 1...

Protein Backbone Dynamics and 15N Chemical Shift Anisotropy from Quantitative Measurement of Relaxation Interference Effects

Cross-correlation between 15N−1H dipolar interactions and 15N chemical shift anisotropy (CSA) gives rise to different relaxation rates of the doublet components of 15N−{1H} peptide backbone amides. A simple scheme for quantitative measurement of this effect is described which yields information on the magnitude of the CSA from the relative intensities of 1H−15N correlations obtained with two slightly different pulse schemes. The method is applied to a sample of uniformly 15N-enriched ubiquitin and measurements conducted at two field strengths (8.5 and 14 T) yield identical results. The degree of relaxation interference correlates with the isotropic 15N chemical shift and results indicate that the sum of the most shielded principal components of the CSA tensor is nearly invariant to structural differences in the polypeptide backbone. The relaxation interference is directly proportional to the generalized order parameter, S2, of the peptide backbone amides, and this relation can be utilized to obtain approximate values for these order parameters.

Magnetic Field Dependence of Nitrogen−Proton J Splittings in 15N-Enriched Human Ubiquitin Resulting from Relaxation Interference and Residual Dipolar Coupling

One-bond 1JNH couplings have been measured in 15N-enriched human ubiquitin and range from 91.1 to 95.6 Hz. Measurements have been carried out using two different methods and at 1H frequencies of 360, 500, and 600 MHz. The best method yields a precision of ca 0.02 Hz, and permits reliable measurement of the small changes (<0.3 Hz) in 1JNH splitting that occur when the magnetic field strength is increased from 8.5 to 14 T. The dependence of the 1JNH splittings on the strength of the static magnetic field originates from two sources: a dynamic frequency shift caused by interference of the 15N chemical shift anisotropy and the 15N−1H dipolar coupling relaxation mechanisms, and a dipolar contribution caused by a small degree of alignment resulting from the anisotropic magnetic susceptibility of the diamagnetic protein. Best fitting of the measured data yields an orientation-independent decrease of 0.11 Hz in the 1JNH splittings at 600 MHz relative to 360 MHz, in perfect agreement with theoretical predictions for the magnitude of the dynamic frequency shift. When fitting the measured J values to the theoretical model, containing only the dynamic frequency shift and dipolar coupling contributions, the reduced error in the statistical F-test is smaller than one, assuming a 0.02 Hz rms error in the experimental 1JNH splittings. This confirms that the random error in the measured data JNH values does not exceed 0.02 Hz, and that effects other than the dipolar coupling and dynamic frequency shift are not detectable. Dependence of the change in 1JNH on the orientation of the N−H bond vector within the molecular frame yields experimentally determined axial and rhombic magnetic shielding susceptibility anisotropies of −2.1 × 10-28 and 0.7 × 10-28 cm3/molecule, respectively. A small improvement of the fit is observed when the amide proton is positioned at a distance above or below the C‘i-1−Ni−Cαi plane which is about five times smaller than the out-of-plane distance predicted by ab initio calculations on a dipeptide analog in vacuum.

A method for studying the structure of uniaxially aligned biopolymers using solid state 15N‐nmr: Application to Bombyx mori silk fibroin fibers

Recent advances in the application of solid state nmr spectroscopy to uniformly aligned biopolymers have opened a window through which to view the detailed structure of biological macromolecules that are unable to be seen with standard techniques for structure determination such as x-ray diffraction. Atomic resolution structural details are obtained from solid state nmr data in the form of bond orientations, which yield the relative positions of specific atoms within the molecule. For static aligned systems such as fibers, in which rapid reorientation about the axis of alignment does not occur, it has generally been necessary to perform trial and error line-shape simulations to extract structural details from nmr spectra arising from a single type of nuclear spin interaction. In the present work, a new method is developed in which solid state 15N-nmr spectra obtained from uniaxially aligned molecules placed with the axis of alignment both parallel and perpendicular to the magnetic field are analyzed to yield the orientations of specific molecular bonds. Analytical expressions are derived that utilize spectral features read from 15N chemical shift anisotropy line shapes to calculate a discrete number of possible orientations for a specific site. The 15N-1H dipolar interaction is employed to further narrow the number of unique orientations possible for a given site. With this method, a neighborhood of possible orientations is quickly determined, and full line-shape simulations within this region of allowed space can be performed to refine the limits of orientation. This technique demonstrates the use of a single type of isotopic label to determine the orientation of a specific molecular group such as a peptide plane within a protein. Results from the application of this method to the Bombyx mori silk fibroin protein provide structural detail that is consistent with currently accepted structural models based on fiber diffraction studies.

Solid state 15N nuclear magnetic resonance of 15N-labeled nylon 6 and nylon 11: Observation of multiple crystalline forms and amorphous regions

The solid state 15N nuclear magnetic resonance (NMR) characterization of nylon 6 and nylon 11 is reported. Nylon 6 (20% 15N enriched) was prepared by anionic polymerization of isotopically enriched caprolactam, and NMR samples were prepared by quenching from the melt, and by slow cooling and annealing. Cross-polarization (CP)-magic-angle-spinning (MAS) spectra of the 15N-enriched samples showed a single sharp peak (α crystal form) at 84.2 ppm (relative to glycine) and a broader resonance at 87.2 ppm. Relaxation experiments were conducted to determine T 1N, T 1H and T 1 ρ for each sample at 300 K. The crystalline resonance was found to have T 1N values of 125–416 s, while the down-field peak had two measurable T 1N values, one component with a T 1 of 1–3 s and a second with the longer T 1 of 19–29 s. The two components of the non-crystalline peak are thought to belong to a liquid-like amorphous region and a more rigid “interphase” region lying between the crystalline and amorphous regions. T 1 ρ measurements were consistent with two-phase morphology although two-component decay for the amorphous region was not observed. 1H T 1 measurements were apparently dominated by spin diffusion that masked any differences between the regions. The chemical shift anisotropy (CSA) of static samples was also obtained. Motion in the amorphous region was monitored by observing an isotropic peak at elevated temperatures. At temperatures above 100 °C, the most deshielded ( σ 33) component was lost from the CSA spectrum, suggesting a previously unreported motion occuring in the rigid crystalline region. This motion is speculated to be associated with the intermolecular hydrogen bond between adjacent chains. Polyundecanamide (nylon 11) was synthesized with 99 + % 15N labeling of the amide nitrogen. Polymer samples were thermally treated to give the stable triclinic α crystal form. The γ form was obtained by precipitation from neat trifluoroacetic acid upon evaporation. 15N CP-MAS NMR clearly differentiated the samples by chemical shift. The α crystal form transformed above 95 °C to the pseudohexagonal δ crystal form, confirming previously reported X-ray and thermal analysis. 15N spin-lattice ( T 1) relaxation experiments confirmed decreased relative mobility in going from the δ to the α form consistent with increased molecular rigidity and density. Previous studies suggested that the α-to-δ transition involves the onset of rapid hydrogen bond disruption and re-formation within the crystal lattice above the 95 °C transition temperature. 15N CSA spectra showed that the hydrogen-bonded amide units remain conformationally rigid in the crystal lattice despite increasing librational motion. Combining 15N T 1 information with wide-angle X-ray data, a model is presented which accounts for the crystal-to-crystal transition.

Nitrogen-15 NMR characterization of multiple crystal forms and phase transitions in polyundecanamide (Nylon 11)

Abstract : Polyundecanamide (nylon 11) was synthesized with 99+% Nitrogen 15 labeling of the amide nitrogen. Polymer samples were thermally treated to give the stable triclinic alpha-crystal form and the metastable delta-smectic form. The gamma-form was obtained by precipitation from neat trifluoroacetic acid upon evaporation. 15N CP/MAS NMR clearly differentiated the samples by chemical shift. Confirming previously reported x-ray and thermal analysis, the alpha- crystal form transforms above 95 C to the pseudohexagonal delta-crystal form. 15N spin lattice (T1) relaxation experiments confirmed decreasing relative mobility in the order delta < delta < alpha consistent with increasing molecular rigidity and density. Previous studies suggested that the alpha-gamma transition involves the onset of rapid hydrogen bond disruption and reformation within the crystal lattice above the 95 C transition temperature. 15N chemical shift anisotropy (CSA) spectra showed that the hydrogen bonded amide units remain conformationally rigid in the crystal lattice despite increasing librational motion. Combining 15N T1 information with wide-angle x-ray data, a model is presented which accounts for the crystal-crystal transition as well as the relative stability of the three crystal forms.

Gramicidin cation channel: an experimental determination of the right-handed helix sense and verification of .beta.-type hydrogen bonding

Due to the difficulty of obtaining protein/lipid cocrystals for diffraction studies, structural research on intrinsic membrane proteins and polypeptides has been largely restricted to indirect experimental techniques. Hence, many fundamental questions associated with peptide/lipid systems remain unanswered. In particular, the handedness of the gramicidin A transmembrane ion channel incorporated into lipid bilayers has been an open question for nearly two decades. In this study, solid-state 15N NMR spectroscopy is employed to probe directly the secondary structure of the polypeptide backbone. Recent determinations of the 15N chemical shift anisotropy tensor with respect to the molecular frame enable the quantitative evaluation of the 15N chemical shift resonances obtained from oriented dimyristoylphosphatidylcholine (DMPC) bilayer samples containing specific site 15N labeled gramicidin. This direct structural approach verifies the beta-sheet hydrogen-bonding pattern proposed by Urry [Urry, D. W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676] and determines that in our DMPC bilayer preparations the gramicidin channel is right-handed. Additional structural information is provided by the 15N chemical shift data in the form of orientational constraints on the C alpha-C alpha axis orientation of individual peptides relative to the helix axis. The significance of these solid-state NMR results lies in the direct determination of the helix sense and the verification of the beta-type hydrogen bonding, in the development of the solid-state NMR methods for obtaining such information, and in emphasizing the importance of having direct structural data at atomic resolution.

Nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin: counterion effects on the 15N shift anisotropy.

High-resolution, solid-state 15N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, we are able to obtain sufficient rotational sideband intensity to determine the full 15N chemical shift anisotropy for the Schiff base nitrogen in bR548 and bR568. Comparisons are made between all-trans-bR568 and N-all-trans-retinylidene butylimine salts with halide, phenolate, and carboxylate counterions. It is argued that for the model compounds the variation in 15N chemical shift reflects the variation in (hydrogen) bond strength with the various counterions. The results suggest that carboxylates and tyrosinates may form hydrogen bonds of comparable strength in a hydrophobic environment. Thus, the hydrogen bonding strength of a counterion depends on factors that are not completely reflected in the solution pKa of its conjugate acid. For the model compounds, the two most downfield principal values of the 15N chemical shift tensor, sigma 22 and sigma 33, vary dramatically with different counterions, whereas sigma 11 remains essentially unaffected. In addition, there exists a linear correlation between sigma 22 and sigma 33, which suggests that a single mechanism is responsible for the variation in chemical shifts present in all three classes of model compounds. The data for bR568 follow this trend, but the isotropic shift is 11 ppm further upfield than any of the model compounds. This extreme value suggests an unusually weak hydrogen bond in the protein.

Dynamics of fd coat protein in the bacteriophage.

The dynamics of the coat protein in fd bacteriophage are described with solid-state 15N and 2H NMR experiments. The virus particles and the coat protein subunits are immobile on the time scales of the 15N chemical shift anisotropy (10(3) Hz) and 2H quadrupole (10(6) Hz) interactions. Previously we have shown that the Trp-26 side chain is immobile, that the two Tyr and three Phe side chains undergo only rapid twofold jump motions about their C beta-C gamma bond axis [Gall, C. M., Cross, T. A., DiVerdi, J. A., & Opella, S. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 101-105], and that most of the backbone peptide linkages are highly constrained but do undergo rapid small amplitude motions [Cross, T. A., & Opella, S. J. (1982) J. Mol. Biol. 159, 543-549] in the coat protein subunits in the virus particles. In this paper, we demonstrate that the four N-terminal residues of the coat protein subunits are highly mobile, since both backbone and side-chain sites of these residues undergo large amplitude motions that are rapid on the time scales of the solid-state NMR experiments. In addition, the dynamics of the methyl-containing aliphatic residues Ala, Leu, Val, Thr, and Met are analyzed. Large amplitude jump motions are observed in nearly all of these side chains even though, with the exception of the N-terminal residue Ala-1, their backbone peptide linkages are highly constrained. The established information about the dynamics of the structural form of fd coat protein in the virus particle is summarized qualitatively.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein dynamics by solid-state nuclear magnetic resonance spectroscopy: Peptide backbone of the coat protein in fd bacteriophage

13C and 15N chemical shift anisotropy and 15N 1H dipolar powder patterns from backbone sites of the coat protein in fd bacteriophage are not averaged by motion. This means that the polypeptide backbone of the protein has no large amplitude motions rapid compared to 10 4 Hz. Relaxation studies on the 13C α and 15N amide resonances indicate the presence of motions on the 10 9 Hz timescale. These results are reconciled with a model where an otherwise rigid backbone undergoes small amplitude, rapid motions.