Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved ¹³C and ¹⁵N signals of [1-¹³C]Tyr-, [¹⁵N]Pro- and [1-¹³C]Val-, [¹⁵N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-¹³C]Val-, [¹⁵N]Pro signals to the specific residues in bR. The previous assignments of [1-¹³C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent ¹⁵N chemical shifts of Pro bonded to Val in the presence of ¹³C-¹⁵N correlation, although no assignment of peak is feasible for ¹³C nuclei not bonded to Pro. ¹³C or ¹⁵N spin-lattice relaxation times (T₁), spin-spin relaxation times under the condition of CP-MAS (T₂), and cross relaxation times (T_CH and T_NH) for ¹³C and ¹⁵N nuclei and carbon or nitrogen-resolved, ¹H spin-lattice relaxation times in the rotating flame (¹H T_1ρ) for the assigned signals were measured in [1-¹³C]Val-, [¹⁵N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from ¹³C and ¹⁵N conformation-dependent chemical shifts and T₁, T₂, ¹H T_1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Proton and phosphorus magnetic relaxation studies on the dynamic structure of single-stranded polyriboadenylic acid

Proton and phosphorus-31 nuclear spin-lattice relaxation times (T₁) have been measured with the Fourier-transform method at 100 and 40.5 MHz, respectively, on single-stranded polyriboadenylic acid (poly(A)) in a neutral D₂O solution in the temperature range of 14–82°C. T₁ minimum is observed around 35–45°C for H(8), H(1′), and phosphorus resonances. Rotational correlation times have been deduced from the T₁ data, which indicate that the sugar–phosphate backbone as well as the base–sugar segment is undergoing rapid internal motion of 10^?8–10^?10 sec range. The molecular motion of the sugar–phosphate backbone as deduced from the phosphorus relaxation is well-characterized by a single activation enthalpy of 8.1 kal/mole for the whole temperature range of 14–82°C. Activation enthalpies of similar magnitude have been obtained for the motion of the adenine–ribose moiety from H(8) and H(1′) relaxation. The relative magnitude of T₁ for H(8) and H(1′) infers that the poly(A) nucleotide exists on the average as anti in the single-stranded form. The phosphorus T₁ value is consistent with a conformation such that both C(4′)–C(5′) and C(4′)–C(3′) bonds are nearly trans to their connected O–P bonds.     

Sampling of Protein Dynamics in Nanosecond Time Scale by 15N NMR Relaxation and Self-Diffusion Measurements

Abstract

This paper presents a procedure for detection of intermediate nanosecond internal dynamics in globular proteins. The procedure uses ¹H-¹⁵N relaxation measurements at several spectrometer frequencies and hydrodynamic calculations based on experimental self-diffusion coefficients. New heteronuclear experiments, using pulse field gradients, are introduced for the measurement of translation diffusion coefficients of ¹⁵N labeled proteins. An advanced interpretation of recently published (Luginbühl et al., Biochemistry, 36, 7305–7312 (1997)) backbone amide ¹⁵N relaxation data, measured at two spectrometers (400 and 750 MHz for ¹H) for N-terminal DNA-binding domain (1–63) of 434 repressor, is presented. Non-applicability of commonly used fast (picosecond) dynamics model (FD) was justified by (i) poor fit of relaxation data by the FD model-free spectral density function both for isotropic and anisotropic models of the overall molecular tumbling; (ii) specific dependence of the overall rotation correlation times calculated from T₁/T₂ ratio on the spectrometer frequency; (iii) mismatch of the ratio of longitudinal ¹⁵N relaxation times T₁, measured at different spectrometer frequencies, in comparison with that anticipated for the IT) model; (iv) significantly underestimated overall rotation correlation time provided by the FD model (5.50±0.15 and 5.80±0.15 ns for 750 and 400 MHz spectrometer frequency respectively) in comparison with correlation time obtained from hydrodynamics. On the other hand, all relaxation and hydrodynamics data are in good correspondence with the model of intermediate (nanoseconds) dynamics. Overall rotation correlation time of 7.5±0.7 ns was calculated from experimental translation self-diffusion rate using hydrodynamics formalism (Garcia de la Torre, J. and Bloomfield, V.A. Quart. Rev. Biophys., 14, 81–139 (1981)). The statistical analysis of ¹⁵N relaxation data along with the hydrodynamic consideration clearly revealed that most of the residues in 434(1–63) repressor are involved in the nanosecond internal dynamics characterized by the the mean order parameters of 0.59±0.06 and the correlation times of ca. 5 ns.     

Protein Rotational Dynamics in Aligned Lipid Membranes Probed by Anisotropic T1ρ NMR Relaxation

A membrane-bound form of Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles was used as a molecular probe to quantify for the viscosity of the lipid membrane interior by measuring the uniaxial rotational diffusion coefficient of the protein. Orientationally dependent ¹⁵N NMR relaxation times in the rotating frame, or T_1ρ, were determined by fitting individually the decay of the resolved NMR peaks corresponding to the transmembrane helix of Pf1 coat protein as a function of the spin-lock time incorporated into the 2D SAMPI4 pulse sequence. The T_1ρ relaxation mechanism was modeled by uniaxial rotational diffusion on a cone, which yields a linear correlation with respect to the bond factor sin⁴θ_B, where θ_B is the angle that the NH bond forms with respect to the axis of rotation. Importantly, the bond factors can be independently measured from the dipolar couplings in the separated local-field SAMPI4 spectra. From this dependence, the value of the diffusion coefficient D_|| = 8.0 × 10⁵ s^?1 was inferred from linear regression of the experimental T_1ρ data even without any spectroscopic assignment. Alternatively, a close value of D_|| = 7.7 × 10⁵ s^?1 was obtained by fitting the T_1ρ relaxation data for the assigned NMR peaks of the transmembrane domain of Pf1 to a wavelike pattern as a function of residue number. The method illustrates the use of single-helix transmembrane peptides as molecular probes to assess the dynamic parameters of biological membranes by NMR relaxation in oriented lipid bilayers.     

Secondary structures of peptides: 5 · 15N n.m.r. CP/MAS spectroscopy of solid polypeptides and gramicidin-S

30.5 MHz ¹⁵N m.m.r. (CP/MAS) spectra of various solid polypeptides were measured using the cross-polarization/magic angle spinning technique. In order to obtain optimum signal-to-noise ratios, relatively short contact times (1 ± 0.5 ms) are required, because the cross-polarization times (T_NH) are short and because the proton rotating-frame relaxation times (T_1p) are in the order of 20 ms. The ¹⁵N n.m.r. signals of copolypeptides may be sensitive to sequence effects; yet they are in most cases more sensitive to the nature of the secondary structure. The signals of α-helices absorb ca. 8–10 ppm upfield of β-sheet structures, whereas the polyglycine II helix absorbs downfield. The natural abundance spectrum of crystalline gramicidin-S exhibits a signal at ?247 ppm, a characteristic chemical shift of the antiparallel pleated sheet structure.     

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the ¹H rotating-frame spin-lattice relaxation (T _1ρ) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by ²H, ¹⁵N and ¹³C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor, amantadine, the protein exhibits significantly narrower linewidths at physiological temperature. We now investigate the origin of this line narrowing through temperature-dependent ¹H T _1ρ relaxation times in the absence and presence of amantadine. Analysis of the temperature dependence indicates that amantadine decreases the correlation time of motion from 2.8 ± 0.9 μs for the apo peptide to 0.89 ± 0.41 μs for the bound peptide at 313 K. Thus the line narrowing of the bound peptide is due to better avoidance of the NMR time scale and suppression of intermediate time scale broadening. The faster diffusion of the bound peptide is due to the higher attempt rate of motion, suggesting that amantadine creates better-packed and more cohesive helical bundles. Analysis of the temperature dependence of $ { \ln }\left( {T_{1\rho }^{ - 1} } \right) $ indicates that the activation energy of motion increased from 14.0 ± 4.0 kJ/mol for the apo peptide to 23.3 ± 6.2 kJ/mol for the bound peptide. This higher activation energy indicates that excess amantadine outside the protein channel in the lipid bilayer increases the membrane viscosity. Thus, the protein-bound amantadine speeds up the diffusion of the helical bundles while the excess amantadine in the bilayer increases the membrane viscosity.     

1H NMRD studies of solutions of paramagnetic metal ions in ethyleneglycol

Solvent¹H T^?1₁ values at magnetic fields between 2.3 × 10^?4 and 1.2 T were measured for ethyleneglycol solutions of manganese(II), cobalt(II), nickel(II), copper(II), and gadolinium(III) salts in the temperature range ?10–+40 °C. The T^?1₁ profiles were interpreted on the ground of the available theories according to whether the electronic relaxation times or the rotational times are the correlation times for the interaction. The comparison among systems experiencing different correlation times and electronic structures (metal ions in water, ethyleneglycol, and in proteins) allowed us to test the general theoretical approach regarding nuclear relaxation times in paramagnetic systems. Such theoretical approaches allow researchers to extract structural parameters and information on the correlation times and dynamic processes operative in the various investigated systems.     

Amide Proton Spin-Lattice Relaxation in Polypeptides: A Field-Dependence Study of the Proton and Nitrogen Dipolar Interactions in Alumichrome

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the ¹H spinlattice relaxation times (T₁'s) are distinct in the Al³⁺-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of ¹H—¹H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T₁^-1 and Σr^-6 at the three magnetic fields. Considering the very sensitive r-dependence of T₁, the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T₁^-1 is proposed to mainly originate from the ¹H-¹⁴N dipolar interaction: this was supported by comparison with measurements on an ¹⁵N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {¹H}—¹H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the ¹H—¹H dipolar mechanism to cause NOE's. From the slope and intersect values of T₁^-1 vs. Σr^-6 linear plots, a number of independent estimates of τ_r, the rotational correlation time, were derived. These and the field-dependence of the T₁'s yield a best estimate <τ_r> ≈ 0.37 ns, in good agreement with 0.38 ns [unk] <τ_r> [unk] 0.41 ns, previously determined from ¹³C and ¹⁵N spin-lattice relaxation data.     

15N spin-lattice relaxation study of linear peptides: Preliminary results on Leu-enkephalin and Tyr-Gly-Gly-Phe

The ability of ¹⁵N relaxation measurements in conformational analysis of linear peptides was studied using Leu-enkephalin: Tyr-Gly-Gly-Phe-Leu and and related tetrapeptide Tyr-Gly-Gly-Phe 95 % ¹⁵N enriched. ¹⁵N spin-lattice relaxation times measured at different temperatures in Me₂SO solution indicate the presence of highly preferential folded structures in both peptides. A marked dependence of T₁ upon the motional effects (segmental rather than anisotropic overall) was observed, while hydrogen bonding affects weakly the relaxation times. From a comparison of ¹⁵N relaxation parameters it appears that the tetrapeptide exhibits a more rigid structure than Leu-enkephalin, in accordance with previous ¹H NMR studies. This paper provides evidence for the usefulness of ¹⁵N T₁ as a mobility probe (independent from ¹³C) in the investigation of the conformational dynamics of peptides.     

Characterization of the backbone dynamics of an anti-digoxin antibody VL domain by inverse detected 1H–15N NMR: Comparisons with X-ray data for the Fab

The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody V_L domain has been characterized by measurements of ¹⁵N T₁ and T₂ relaxation times, ¹H–¹⁵N NOE values, and ¹H–²H exchange rates. These data were acquired with 2D inverse detected heteronuclear ¹H–¹⁵N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R₂ (= 1/T₂). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R₂ exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R₂ exchange contributions. The measured ¹H–²H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the V_L domain, including the three hypervariable loops, undergo dynamic changes upon V_L:V_H association and/ or complexation with digoxin. Overall, the 26–10 V_L domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

Dynamic Structure of Proteins in Solid State. 1H and 13C NMR Relaxation Study

Abstract

Temperature dependencies of ¹H non-selective NMR T₁ and T₂ relaxation times measured at two resonance frequencies and natural abundance ^l3C NMR relaxation times T_l and T_lr measured at room temperature have been studied in a set of dry and wet solid proteins—;Bacterial RNase, lysozyme and Bovine serum albumin (BSA). The proton and carbon data were interpreted in terms of a model supposing three kinds of internal motions in a protein. These are rotation of the methyl protons around the axis of symmetry of the methyl group, and fast and slow oscillations of all atoms. The correlation times of these motions in solid state are found around 10^?11, 10^?9 and 10^?6 s, respectively. All kinds of motion are characterized by the inhomogeneous distribution of the correlation times. The protein dehydration affects only the slow internal motion. The amplitude of the slow motion obtained from the carbon data is substantially less than that obtained from the proton data. This difference can be explained by taking into account different relative inter- and intra- chemical group contributions to the proton and carbon second moments. The comparison of the solid state and solution proton relaxation data showed that the internal protein dynamics in these states is different: the slow motion seems to be few orders of magnitude faster in solution.     

Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T₁) and spin-spin (T₂) relaxation times of water in cysts hydrated with D₂O have been measured for hydrations between 1.5 and 0.1 g of D₂O per gram of dry solids. When the relaxation rates (I/T₁, I/T₂) of ²H and ¹⁷O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D₂O (or H₂ ¹⁷O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The ²H and ¹⁷O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.

It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H₂O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T₁ as a function of hydration in the vicinity of 0.6 g H₂O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

TROSY pulse sequence for simultaneous measurement of the <Superscript>15</Superscript>N <Emphasis Type="Italic">R</Emphasis><Subscript>1</Subscript> and {<Superscript>1</Superscript>H}–<Superscript>15</Superscript>N NOE in deuterated proteins

A TROSY-based NMR experiment is described for simultaneous measurement of the ¹⁵N longitudinal relaxation rate constant R₁ and the {¹H}–¹⁵N nuclear Overhauser enhancement. The experiment is based on the observation that the TROSY mixing pulse sequence element symmetrically exchanges ¹H and ¹⁵N magnetizations. The accuracy of the proposed technique is validated by comparison to independent measurements of both relaxation parameters for the protein ubiquitin. The simultaneous experiment is approximately 20–33% shorter than conventional sequential measurements.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

The conformation of oxytocin in dimethylsulfoxide as revealed by carbon-13 spin-lattice relaxation times

Information was obtained on rates of overall molecular reorientation and segmental motion of amino acid sidechains of oxytocin in dimethylsulfoxide by determination of spin-lattice relaxation times (T₁) at 25 MHz for carbon-13 in natural abundance in the hormone. The T₁ values of the α-carbons of amino acid residues located in the 20-membered ring of oxytocin are all about 50 msec. The overall correlation time for the hormone backbone was estimated to be 8.8 × 10^?10 sec. The sidechains of Tyr, Ile and Gln undergo segmental motion with respect to the backbone of the ring. The T₁ value of the α-carbon of the Leu residue is greater than for any α-carbon in the ring, indicating an increased mobility of the backbone of the C-terminal acyclic peptide as compared to the ring. The β- and γ-carbons of the Pro residue undergo an exo-endo interconversion with regard to the plane formed by α-carbon, δ-carbon and N atom of the Pro pyrollidine ring. These data are discussed in light of results from other experimental and theoretical studies, including carbon-13 spin-lattice relaxation times for oxytocin in aqueous solution.     

Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual ¹⁵N–T ₁ timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s⁻¹. Backbone amide ¹⁵N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D₂O is employed as a solvent for sample preparation. Due to the intrinsically long ¹⁵N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.     

Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields

Summary ¹⁵N NMR relaxation times in perdeuterated HIV-1 protease, complexed with the sub-nanomolar inhibitor DMP323, have been measured at 600 and 360 MHz ¹H frequency. The relative magnitudes of the principal components of the inertia tensor, calculated from the X-ray coordinates of the protein-drug complex, are 1.0:0.85:0.44. The relation between the T₁/T₂ ratios observed for the individual backbone amides and their N-H orientation within the 3D structure of the protease dimer yields a rotational diffusion tensor oriented nearly collinear to the inertia tensor. The relative magnitudes of its principal components (1.00:1.11:1.42) are also in good agreement with hydrodynamic modeling results. The orientation and magnitude of the diffusion tensors derived from relaxation data obtained at 360 and 600 MHz are nearly identical. The anisotropic nature of the rotational diffusion has little influence on the order parameters derived from the ¹⁵N T₁ and T₂ relaxation times; however, if anisotropy is ignored, this can result in erroneous identification of either exchange broadening or internal motions on a nanosecond time scale. The average ratio of the T₁ values measured at 360 and 600 MHz is 0.50±0.015, which is slightly larger than the value of 0.466 expected for an isotropic rigid rotor with _c = 10.7 ns. The average ratio of the T₂ values measured at 360 and 600 MHz is 1.14±0.04, which is also slightly larger than the expected ratio of 1.11. This magnetic field dependence of the T₁ and T₂ relaxation times suggests that the spectral density contribution from fast internal motions is not negligible, and that the chemical shift anisotropy of peptide backbone amides, on average, is larger than the 160 ppm value commonly used in ¹⁵N relaxation studies of proteins.