TROSY NMR with partially deuterated proteins

TROSY-type optimization of liquid-state NMR experiments is based on the preservation of unique coherence transfer pathways with distinct transverse relaxation properties. The broadband decoupling of the ¹H spins interchanges the TROSY and anti-TROSY magnetization transfer pathways and thus is not used in TROSY-type triple resonance experiments or is replaced with narrowband selective decoupling. To achieve the full advantage of TROSY, the uniform deuteration of proteins is usually required. Here we propose a new and general method for ¹H broadband decoupling in TROSY NMR, which does not compromise the relaxation optimization in the ¹⁵N–¹H moieties, but uniformly and efficiently refocuses the ¹ J _CH scalar coupling evolution in the ¹³C–¹H moieties. Combined with the conventional ²H decoupling, this method enables obtaining high sensitivity TROSY-type triple resonance spectra with partially deuterated or fully protonated ¹³C,¹⁵N labeled proteins.     

Improved TROSY-HNCA experiment with suppression of conformational exchange induced relaxation

A general method for improving of the sensitivity of the TROSY-type triple resonance experiments in the presence of conformational exchange-induced (CSX) relaxation is proposed based on the use of CPMG-INEPT (Müller et al., J. Am. Chem. Soc., 1995, 117, 11043–11048) during the N–C polarization transfer periods. Significantly improved sensitivity is demonstrated for the majority of cross-peaks in the new [¹⁵N,¹H]-TROSY-XY-HNCA experiment, measured with partially folded RNase AS-Protein, with negligible loss of sensitivity for resonances unaffected by CSX relaxation. In addition, a comparison of cross-peak amplitudes in [¹⁵N,¹N]-TROSY-XY-HNCA and conventional [¹⁵N,¹H]-TROSY-HNCA spectra provides a quick and sensitive estimation of the CSX relaxation contribution.     

A TROSY CPMG sequence for characterizing chemical exchange in large proteins

A new NMR spin relaxation experiment is described for measuring chemical exchange time constants from approximately 0.5 ms to 5 ms in ¹⁵N-labeled macromolecules. The pulse sequence is based on the Carr–Purcell–Meiboom–Gill technique [Carr and Purcell (1954) Phys. Rev., 94, 630–638; Meiboom and Gill (1958) Rev. Sci. Instrum., 29, 688–691; Loria et al. (1999) J. Am. Chem. Soc., 121, 2331–2332], but implements TROSY selection [Pervushin et al. (1997) Proc. Natl. Acad. Sci. USA, 94, 12366–12371] to permit measurement of exchange linebroadening contributions to the narrower component of the ¹H-¹⁵N scalar-coupled doublet. This modification extends the size limitation imposed on relaxation measurements due to the fast decay of transverse magnetization in larger macromolecules. The new TROSY-CPMG experiment is demonstrated on a [U-98% ¹⁵ N] labeled sample of basic pancreatic trypsin inhibitor and a [U-83% ²H, U-98% ¹⁵ N] labeled sample of triosephosphate isomerase, a 54 kDa homodimeric protein.     

TROSY in NMR studies of the structure and function of large biological macromolecules

Transverse relaxation-optimized spectroscopy (TROSY), in combination with various isotope-labeling techniques, has opened avenues to study biomolecules with molecular masses of up to 1000000Da by solution NMR. Important recent applications of TROSY include the structure determination of membrane proteins in detergent micelles, structural and functional studies of large proteins in both monomeric form and macromolecular complexes, and investigations of intermolecular interactions in large complexes. TROSY improves the measurement of residual dipolar couplings and the detection of scalar couplings across hydrogen bonds - techniques that promise to further enhance the determination of solution structures of large proteins and oligonucleotides.     

Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium

In this paper it is demonstrated that cross-correlated time modulation of isotropic chemical shifts (`conformational exchange') leads to differential relaxation of double- and zero-quantum coherences, respectively. Quantitative information can be obtained from the time dependence of the interconversion between the two two-spin coherences 2I_xS_x and 2I_yS_y, induced by the differential relaxation. The effect is illustrated with an application to ¹³C,¹⁵N-labeled quail CRP2(LIM2), by studying ¹⁵N-¹H^N multiple-quantum relaxation. Significant cross-correlated fluctuations of isotropic chemical shifts were observed for residues which are part of a disordered loop region connecting two -strands in CRP2(LIM2). Differential ¹H^N and ¹⁵N exchange contributions to multiple-quantum relaxation observed at these sites illustrate the complex interplay between hydrogen bonding events and conformational reorientations in proteins.     

NMR detection of multiple transitions to low-populated states in azurin

Transitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone (15)N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8 degrees C). Two exchange processes with different rate constants could be discriminated despite populations of the excited states below 1% and spatial neighborhood of the two processes. The group of (15)N nuclei involved in the faster process exhibits at 44.8 degrees C a forward rate constant of 11.7+/-2.4 s(-1) and a population of the exited state of 0.39+/-0.07%. They surround the aromatic ring of histidine 35 whose protonation state is coupled to the flipping of a neighboring peptide plane. For the slower process, the forward rate constant and population of the exited state at 44.8 degrees C are 4.1+/-0.1 s(-1) and 0.45+/-0.02%, respectively. The residues involved cluster nearby the copper ion, which is separated from the protonation site of histidine 35 by about 8 A, indicating conformational rearrangements involving the copper coordinating loops. The dependence of the equilibrium constant on the temperature is consistent with an enthalpy-dominated transition around the copper, but an entropy-controlled transition near histidine 35. The detection by nuclear magnetic resonance of millisecond to second conformational transitions near the copper ion suggests a low energy-cost rearrangement of the copper-binding site that may be necessary for efficient electron transfer.     

Sensitivity enhancement in NMR of macromolecules by application of optimal control theory

NMR of macromolecules is limited by large transverse relaxation rates. In practice, this results in low efficiency of coherence transfer steps in multidimensional NMR experiments, leading to poor sensitivity and long acquisition times. The efficiency of coherence transfer can be maximized by design of relaxation optimized pulse sequences using tools from optimal control theory. In this paper, we demonstrate that this approach can be adopted for studies of large biological systems, such as the 800 kDa chaperone GroEL. For this system, the ¹H–¹⁵N coherence transfer module presented here yields an average sensitivity enhancement of 20–25% for cross-correlated relaxation induced polarization transfer (CRIPT) experiments.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-3592-0     

Enhancement of the steady-state magnetization in TROSY experiments

Under the condition that the longitudinal relaxation time of spin I is shorter than the longitudinal relaxation time of spin S the steady-state magnetization in [S,I]-TROSY-type experiments can be enhanced by intermediate storage of a part of the steady-state magnetization of spin I on spin S with a pulse sequence element during the relaxation delay. It is demonstrated with samples ranging in size from the 1 kDa cyclosporin to the 110 kDa ¹⁵N,²H-labeled dihydroneopterin Aldolase that intermediate storage of steady-state magnetization in a [¹⁵N,¹H]-TROSY experiment yields a signal gain of 10–25%. The method proposed here for intermediate storage of steady-state magnetization can be implemented in any [¹⁵N,¹H]-TROSY-type experiments.     

Microsecond timescale backbone conformational dynamics in ubiquitin studied with NMR R1rho relaxation experiments

NMR spin relaxation experiments are used to characterize the dynamics of the backbone of ubiquitin. Chemical exchange processes affecting residues Ile 23, Asn 25, Thr 55, and Val 70 are characterized using on- and off-resonance rotating-frame 15N R1rho relaxation experiments to have a kinetic exchange rate constant of 25,000 sec(-1) at 280 K. The exchange process affecting residues 23, 25, and 55 appears to result from disruption of N-cap hydrogen bonds of the alpha-helix and possibly from repacking of the side chain of Ile 23. Chemical exchange processes affecting other residues on the surface of ubiquitin are identified using 1H-15N multiple quantum relaxation experiments. These residues are located near or at the regions known to interact with various enzymes of the ubiquitin-dependent protein degradation pathway.     

NMR detection of slow conformational dynamics in an endonuclease toxin

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid¹⁵ N- and ¹³C/¹⁵N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro⁶⁵-Asn⁷², which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D ¹H-¹H-¹⁵N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with C and C chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp²² or Trp⁵⁸, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D¹ H-¹H-¹⁵N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, N_A and N_B, which interchange at 15 °C with a forward rate constant of 1.61 ± 0.5 s^-1 and a backward rate constant of 1.05 ± 0.5 s^-1. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743–13750 and 13751–13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the N_A and N_B conformers of the DNase and with binding only to the N_B conformer detected because the rate of dissociation of the complex of Im9 and the N_A conformer, N_AI, is extremely rapid. In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the N_BI form. The possibility that cis–trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed. E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the N_A N_B interconversion to be identified, Pro⁶⁵ and Pro⁷³. The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form.     

NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.

Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two- and three-dimensional NMR. Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain. The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout.     

Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures

Dynamic properties of electron transfer pathways in a small blue copper cupredoxin are explored using an extensive 15N NMR relaxation study of reduced Pseudomonas aeruginosa azurin at four magnetic fields (500-900 MHz) and at two temperatures chosen well below the melting point of the protein. Following a careful model-free analysis, several protein regions with different dynamic regimes are identified. Nanosecond time-scale mobility characterizes various residues of the hydrophobic surface patch believed to mark the natural entry point for electrons, notably the surface-exposed copper-ligand His117. These findings are consistent with a gated electron transfer process according to the "dynamic docking" model. Residues 47-49 along intramolecular pathways of electrons show rigidity that is remarkably conserved when increasing the temperature. Three different conformational exchange processes were observed in the millisecond range, one near the only disulfide bridge in the molecule and two near the copper ion. The latter two processes are consistent with previous data such as crystal structures at various pH values and NMR relaxation dispersion experiments; they may indicate an additional gated electron transfer mechanism at slower time-scales.     

NMR exchange broadening arising from specific low affinity protein self-association: Analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1

Nuclear spin relaxation monitored by heteronuclear NMR provides a useful method to probe the overall and internal molecular motion for biological macromolecules over a variety of time scales. Nitrogen-15 NMR relaxation parameters have been recorded for the N-terminal domain of the rat T-cell antigen CD2 (CD2d1) in a dilution series from 1.20 mM to 40 M (pH 6.0, 25 °C). The data have been analysed within the framework of the model- free formalism of Lipari and Szabo to understand the molecular origin of severely enhanced transverse relaxation rates found for certain residues. These data revealed a strong dependence of the derived molecular correlation time _c upon the CD2d1 protein concentration. Moreover, a number of amide NH resonances exhibited exchange broadening and chemical shifts both strongly dependent on protein concentration. These amide groups cluster on the major -sheet surface of CD2d1 that coincides with a major lattice contact in the X-ray structure of the intact ectodomain of rat CD2. The complete set of relaxation data fit well to an equilibrium monomer–dimer exchange model, yielding estimates of exchange rate constants (k_ON=5000 M^-1 s^-1; k_OFF=7 s^-1) and a dissociation constant (K_D 3–6 mM) that is consistent with the difficulty in detecting the weak interactions for this molecule by alternative biophysical methods. The self-association of CD2d1 is essentially invariant to changes in buffer composition and ionic strength and the associated relaxation phenomena cannot be explained as a result of neglecting anisotropic rotational diffusion in the analysis. These observations highlight the necessity to consider low affinity protein self-association interactions as a source of residue specific exchange phenomena in NMR spectra of macromolecular biomolecules, before the assignment of more elaborate intramolecular conformational mechanisms.     

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole–dipole interactions and the associated “spin diffusion” due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30 kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experiment.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-3070-8     

The use of spin desalting columns in DMSO‐quenched H/D‐exchange NMR experiments

Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D₂O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D₂O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride.     

Application of NMR,molecular simulation,and hydrodynamics to conformational analysis of trisaccharides

The preferred conformations and conformational flexibilities of the trisaccharides alpha-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe (I) and alpha-D-Glcp-(1-->3)[beta-D-Glcp-(1-->4)]-alpha-D-Glcp-OMe (II) in aqueous solution were determined using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics (MD) and Langevin dynamics (LD) simulations, and hydrodynamics calculations. Both trisaccharides have a vicinal substitution pattern in which long range (nonsequential) interactions may play an important role. LD simulation at 600 K indicated that the all-syn conformation predominated, though other conformations were apparent. NOE data and MD and LD simulations at 298 K all indicated that trisaccharide I is a single all-syn conformer in solution. Given that previous studies showed evidence of anti-conformers in beta-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe, this result provides an example of how changing the anomeric configuration of one residue from beta to alpha can make an oligosaccharide more rigid. Discrepancies in inter-ring distances obtained by experiment and by simulation of the all-syn conformer suggest the presence of an anti-psi conformation at the beta-(1-->4)-linkage for II. A combined analysis of measured and calculated translational diffusion constants and (13)C T(1) relaxation times yield order parameters of 0.9 for each trisaccharide. This implies that any interconversion among conformations is significantly slower than tumbling. Anisotropies of approximately 1.6 and 1.3 calculated for I and II, respectively, are consistent with the observed relatively flat T(1) profiles because the tumbling is not in the motional narrowing regime.     

Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

NMR spin relaxation in the rotating frame (R_1ρ) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide ¹H off-resonance R_1ρ relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using ¹⁵N or ¹³C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The ¹H off-resonance R_1ρ data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 μs. This is in contrast to the case for ¹⁵N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly ¹⁵N-enriched samples that were either highly enriched in ²H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the ¹H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present ¹H off-resonance R_1ρ experiments are in excellent agreement with those determined previously using a combination of ¹⁵N laboratory-frame and off-resonance R_1ρ relaxation data, with average values of and 21 ± 3 μs, respectively.     

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/D–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/D1.5, 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.     

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.     

Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G

Because the overall tumbling provides a major contribution to protein spectral densities measured in solution, the choice of a proper model for this motion is critical for accurate analysis of protein dynamics. Here we study the overall and backbone dynamics of the B3 domain of protein G using ¹⁵N relaxation measurements and show that the picture of local motions is markedly dependent on the model of overall tumbling. The main difference is in the interpretation of the elevated R ₂ values in the -helix: the isotropic model results in conformational exchange throughout the entire helix, whereas no exchange is predicted by anisotropic models that place the longitudinal axis of diffusion tensor almost parallel to the helix axis. Due to small size (fast tumbling) of the protein, the T ₁ values have low sensitivity to NH bond orientation. The diffusion tensor derived from orientation dependence of R ₂/R ₁ is anisotropic (D _par/D _perp=1.4), with a small rhombic component. In order to distinguish the correct picture of motion, we apply model-independent methods that are sensitive to conformational exchange and do not require knowledge of protein structure or assumptions about its dynamics. A comparison of the CSA/dipolar cross-correlation rate constants with ¹⁵N relaxation rates and the estimation of R _ex terms from relaxation data at 9.4 and 14.1 T indicate no conformational exchange in the helix, in support of the anisotropic models. The experimentally derived diffusion tensor is in excellent agreement with theoretical predictions from hydrodynamic calculations; a detailed comparison with various hydrodynamic models revealed optimal parameters for hydrodynamic calculations.