Deuterium Spin Relaxation Research Articles

Proton and deuterium nuclear spin relaxation study of the SmA and SmC* phases of BP8Cl-d17 : a self-consistent analysis.

A self-consistent analysis of proton and deuterium nuclear spin relaxation times in the smectic phases of a partially deuterated smectogen is presented here. Proton spin-lattice relaxation times T(1Z) were measured as a function of Larmor frequency over a range of 1 kHz to 300 MHz at selected temperatures. Deuterium spin relaxation times T(1Z) and T(1Q) were measured as a function of temperature at two different magnetic fields in the smectic A phase. The deuterium data provide dynamic parameters such as rotational diffusion constants and internal jump rates as well as the nematic order parameter S. The proton data are analyzed using a number of relaxation mechanisms, one of which is the molecular reorientation. It is found helpful in these latter analyses to use the nematic order parameter and to fix the contribution from molecular reorientations determined by the deuterium spin relaxation. The fits to the proton T(1) frequency and temperature dispersions by the remaining relaxation mechanisms such as layer undulations and translational self-diffusion will be discussed for the smectic A and chiral smectic C phases.

Is Buffer a Good Proxy for a Crowded Cell-Like Environment? A Comparative NMR Study of Calmodulin Side-Chain Dynamics in Buffer and E. coli Lysate

Biophysical studies of protein structure and dynamics are typically performed in a highly controlled manner involving only the protein(s) of interest. Comparatively fewer such studies have been carried out in the context of a cellular environment that typically involves many biomolecules, ions and metabolites. Recently, solution NMR spectroscopy, focusing primarily on backbone amide groups as reporters, has emerged as a powerful technique for investigating protein structure and dynamics in vivo and in crowded “cell-like” environments. Here we extend these studies through a comparative analysis of Ile, Leu, Val and Met methyl side-chain motions in apo, Ca2+-bound and Ca2+, peptide-bound calmodulin dissolved in aqueous buffer or in E. coli lysate. Deuterium spin relaxation experiments, sensitive to pico- to nano-second time-scale processes and Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, reporting on millisecond dynamics, have been recorded. Both similarities and differences in motional properties are noted for calmodulin dissolved in buffer or in lysate. These results emphasize that while significant insights can be obtained through detailed “test-tube” studies, experiments performed under conditions that are “cell-like” are critical for obtaining a comprehensive understanding of protein motion in vivo and therefore for elucidating the relation between motion and function.

Probing Side-Chain Dynamics in Proteins by the Measurement of Nine Deuterium Relaxation Rates Per Methyl Group

We demonstrate the feasibility of the measurement of up to nine deuterium spin relaxation rates in 13CHD2 and 13CH2D methyl isotopomers of small proteins. In addition to five measurable 2H relaxation rates in a 13CH2D methyl group (Millet, O.; Muhandiram, D. R.; Skrynnikov, N. R.; Kay, L. E. J. Am. Chem. Soc. 2002, 124, 6439-48), the measurement of additional four rates of (nearly) single-exponentially decaying magnetization terms in methyl groups of the 13CHD2 variety is reported. Consistency relationships between 2H spin relaxation rates measured in the two different types of methyl groups are derived and verified experimentally for a subset of methyl-containing side chains in the protein ubiquitin. A detailed comparison of methyl-bearing side-chain dynamics parameters obtained from relaxation measurements in 13CH2D and 13CHD2 methyls of ubiquitin at 10, 27, and 40 °C reveals that transverse 2H relaxation rates in 13CHD2 groups are reliable and accurate reporters of the amplitudes of methyl 3-fold axis motions (S(axis)2) for protein molecules with global molecular tumbling times τ(C) >~9 ns. For smaller molecules, simple correction of transverse 2H relaxation rates in 13CHD2 groups is sufficient for the derivation of robust measures of order. Residue-specific distributions of S(axis)2 are consistent with atomic-detail molecular dynamics (MD) results. Both 13CHD2- and 13CH2D-derived S(axis)2 values are in good overall agreement with those obtained from 1 μs MD simulations at all the three temperatures, although some differences in the site-specific temperature dependence between MD- and 2H-relaxation-derived S(axis)2 values are observed.

A tribute to Lewis E Kay on his 50th birthday

It is a great pleasure for us to introduce this special issue of the Journal of Biomolecular NMR on the occasion of Prof. Lewis E. Kay’s 50th birthday. We are grateful to the Editors of the Journal of Biomolecular NMR for accepting our proposal. In particular, we thank Prof. Martin Billeter for editing this issue, and Dr. Ad Bax for advice and support. We believe that Lewis merits this special issue of Journal of Biomolecular NMR for his innovative contributions to biomolecular NMR spectroscopy, as well as for his role as inspirer for a large group of young research professionals. The following short list of examples serves to illustrate how the work of Lewis Kay has helped to advance our field: Lewis has consistently been a pioneer in the development of novel methods essential to increasing the size of proteins that may be studied by NMR. In the days when assigning chemical shifts of a protein with 150 amino acids was a major endeavour, the Kay lab––in collaboration with Prof. Cheryl Arrowsmith––combined advances in gradientenhanced spectroscopy, protein deuteration and pulse sequence design to establish the resonance assignments for a 37 kDa complex. As these techniques became more broadly used, his lab forged ahead with clever biochemical tricks for incorporating protonated methyl groups in otherwise deuterated proteins, and developed methyl TROSY experiments that permit well-resolved spectra to be obtained for high molecular weight complexes, extending to 1 MDa. Lewis has been a leader in using NMR to measure fast timescale macromolecular dynamics, starting with his contributions to inverse-detected N relaxation experiments. His deuterium spin relaxation methodologies have had a huge impact on our ability to characterize side chain dynamics in proteins. In addition, the Kay lab has extensively developed and applied new methodologies for studying low-populated states of proteins and their roles in biology. Building on transverse relaxation dispersion experiments, the Kay lab has introduced approaches to elucidate the threeK. H. Gardner (&) Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8816, USA e-mail: Kevin.Gardner@UTSouthwestern.edu

Water dynamics and salt-activation of enzymes in organic media: mechanistic implications revealed by NMR spectroscopy.

Deuterium spin relaxation was used to examine the motion of enzyme-bound water on subtilisin Carlsberg co-lyophilized with inorganic salts for activation in different organic solvents. Spectral editing was used to ensure that the relaxation times were associated with relatively mobile deuterons, which were contributed almost entirely by D(2)O rather than hydrogen-deuteron exchange on the protein. The results indicate that the timescale of motion for residual water molecules on the biocatalyst, (tau(c))(D(2)O), in hexane decreased from 65 ns (salt-free) to 0.58 ns (98% CsF) as (k(cat)/K(M))(app) of the biocatalyst preparation increased from 0.092 s(-1) x M(-1) (salt-free) to 1,140 s(-1) x M(-1) (98% CsF). A similar effect was apparent in acetone; the timescale decreased from 24 ns (salt-free) to 2.87 ns (98% KF), with a corresponding increase in (k(cat)/K(M))(app) of 0.140 s(-1) x M(-1) (salt-free) to 12.8 s(-1) x M(-1) (98% KF). Although a global correlation between water mobility and enzyme activity was not evident, linear correlations between ln[(k(cat)/K(M))(app)] and (tau(c))(D(2)O) were obtained for salt-activated enzyme preparations in both hexane and acetone. Furthermore, a direct correlation was evident between (k(cat)/K(M))(app) and the total amount of mobile water per mass of enzyme. These results suggest that increases in enzyme-bound water mobility mediated by the presence of salt act as a molecular lubricant and enhance enzyme flexibility in a manner functionally similar to temperature. Greater flexibility may permit a larger degree of local transition-state mobility, reflected by a more positive entropy of activation, for the salt-activated enzyme compared with the salt-free enzyme. This increased mobility may contribute to the dramatic increases in biocatalyst activity.

NMR Dynamics-Derived Insights into the Binding Properties of a Peptide Interacting with an SH2 Domain

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.

Side chain dynamics in unfolded protein states: an NMR based 2H spin relaxation study of delta131delta.

NMR relaxation data on disordered proteins can provide insight into both structural and dynamic properties of these molecules. Because of chemical shift degeneracy in correlation spectra, detailed site-specific analyses of side chain dynamics have not been possible. Here, we present new experiments for the measurement of side chain dynamics in methyl-containing residues in unfolded protein states. The pulse schemes are similar to recently proposed methods for measuring deuterium spin relaxation rates in (13)CH(2)D methyl groups in folded proteins.(1) However, because resolution in (1)H-(13)C correlation maps of unfolded proteins is limiting, relaxation data are recorded as a series of (1)H-(15)N spectra. The methodology is illustrated with an application to the study of side chain dynamics in delta131delta, a large disordered fragment of staphylococcal nuclease containing residues 1-3 and 13-140 of the wide-type protein. A good correlation between the order parameters of the symmetry axes of the methyl groups and the backbone (1)H-(15)N bond vectors of the same residue is observed. Simulations establish that such a correlation is only possible if the unfolded state is comprised of an ensemble of structures which are not equiprobable. A motional model, which combines wobbling-in-a-cone and Gaussian axial fluctuations, is proposed to estimate chi(1) torsion angle fluctuations, sigma(chi)()1, of Val and Thr residues on the basis of the backbone and side chain order parameters. Values of sigma(chi)()1 are approximately 10 degrees larger than what has previously been observed in folded proteins. Of interest, the value of sigma(chi)()1 for Val 104 is considerably smaller than for other Val or Thr residues, suggesting that it may be part of a hydrophobic cluster. Notably large (15)N transverse relaxation rates are observed in this region. To our knowledge, this is the first time that side chain dynamics in an unfolded state have been studied in detail by NMR.

Dynamics of a Liquid Crystal in its Smectic A Phase from Angle Dependent Deuterium Spin Relaxation Measurements

The Zeeman and quadrupolar spin-lattice relaxation times of the aromatic deuterons of OAB-d 12 were measured throughout the smectic A phase at two different frequencies (15 and 46.04 MHz). At 15 MHz measurements were performed as a function of the angle between the phase director and the external magnetic field. Eight spectral densities were consequently determined, and were analyzed by means of a global target fitting procedure using diffusional models for the overall molecular reorientations and the internal motions. Diffusional coefficients of about 10 9 s −1 were found for molecular spinning and phenyl ring rotations, three orders of magnitude higher than for molecular tumbling.

The fluidity of hydrocarbon regions in organo-gels, studied by NMR: Basic translational and rotational diffusion measurements

Organogels composed of 12-hydroxystearic acid (12-HSA) and low viscosity solvents such as decane, decalin and ethyl acetate have been used as model systems for lubricating greases. The fluidity of perdeuterated probe molecules in the above mentioned model systems was studied using two NMR spectroscopy methods. First, translational diffusion was studied for the perdeuterated probes benzene, toluene, decane and dodecane by pulsed-field gradient NMR. The probes were introduced into a commercial lubricating grease and the model systems mentioned above. Second, the rotational diffusion tensor for perdeuterated trans-decalin was studied by deuterium spin relaxation measurements. All probed samples contained 5% probe (by total mass). The measurements show that the microviscosity in the gels is quite similar to the bulk viscosity in the corresponding neat solvent. This indicates that no strong interactions occur between 12-HSA, and it also indicates that the obstruction effect is responsible for the decrease in diffusion rate compared to the neat solvents in 12-HSA-thickened systems.

Individual spectral densities and molecular motion in polycrystalline hexamethylbenzene-d18

Methods are described for obtaining the orientation dependence of individual motional spectral densities, J1(ω0) and J2(2ω0), from deuterium spin relaxation experiments on polycrystalline materials. Spectral density measurements provide detailed information in a motional regime too fast to be studied by the two-dimensional (2D) exchange method. Their potential as a source of detailed kinetic and geometric information is illustrated for hexamethylbenzene-d18 (HMB). The relaxation behavior of HMB cannot be explained exclusively by six-site jumps around the C6v axis. Agreement between the experimentally determined spectral densities and simulations is improved if the methyl rotation is explicitly included. At ambient temperature the experimental data are best fitted with the simultaneous jump rates, k6=3.85×108 s−1 and k3=5.0×1011 s−1. This is significantly different from the rate determined using a simple six-site jump model, k6=3.9×109 s−1. Geometric distortions of the methyl rotation axes can account for the observed motionally averaged electric field gradient tensor. When these distortions are included in analysis of the spectral density data, there is a small, but significant, improvement in the fit. k3 is unchanged and the best fit k6 is reduced to 2.2×108 s−1, with distortions out of plane by δ=2.5° and in plane ε=ε′=1.202.

Deuterium spin relaxation and molecular motion in a binary liquid-crystal mixture

Deuterium spin relaxation has been studied as a function of temperature, concentration, and site of deuteration for both components of a binary nematic liquid-crystal mixture of 4-methyl-4′-cyanobiphenyl- d 11 (1CB) and 4- n-pentyl-4′-cyanobiphenyl- d 6 (5CB). Relaxation rates of Zeeman order R 1Z and quadrupolar order R 1Q were determined by two different techniques. It was found that the standard method, based on sum and difference (SAD) magnetization generated by a Jeener-Broekaert sequence, suffers from serious systematic error due to frequency-selective excitation, large linear phase corrections, and unavoidable baseline distortion. Broadband Jeener-Broekaert (BBJB) sequences can be used to achieve uniform excitation of quadrupole order, while echo techniques can be used both with inversion-recovery (IR) and BBJB sequences to eliminate baseline artifacts. This new approach of performing two separate experiments (IR/BBJB) yields spectral densities J 1( ω 0) and J 2(2 ω 0) with precision and accuracy significantly greater than those determined by the SAD method. For 1CB, the SAD results were not accurate enough for detailed analysis but a satisfactory interpretation of the temperature-dependent IR/BBJB data for 1 CB methyl and aromatic deuterons was achieved in terms of a model with three correlation times. The same model was successfully used to account for relaxation of the alkyl chain C, deuterons in the liquid-crystal molecule 5CB. Measurements on 10 and 25 mol% mixtures showed that the correlation times and activation energies for rotational motion of both 1CB and 5CB are concentration independent.

Molecular dynamics in a nematogen studied by deuterium nuclear magnetic resonance spectroscopy

The deuterium spin relaxation times of the nematic liquid crystal p-[2H3]methoxy[2H1]benzylidene-p-n-[2H9]butylaniline ([2H13]MBBA) have been determined using the phase-cycled Jeener–Broekaert pulse sequence. The spectral densities of motion of the methine and chain deuterons have been derived; they contain information on the overall and internal motions of the molecules. The overall motion is discussed in terms of the small-step rotational diffusion model, and the segmental motion of the butyl chain is considered using the superimposed free rotations model. In addition, the possible contribution of director fluctuations to the relaxation of deuteron spins is considered. The computed values of the various rotational diffusion coefficients are given. When the effects of director fluctuations are included the results are generally physically reasonable. One interesting point is that the contribution to spin relaxation due to director fluctuations appears to show an odd–even effect along the butyl chain of MBBA.

Spectral Density of Motion in a Reentrant Nematic by Deuterium NMR

Abstract We report on a deuterium spin relaxation study of a 26 wt. % sample of perdeuterated hexyloxycyanobiphenyl (60CB-d 21) in octyloxycyanobiphenyl as a function of temperature at a Larmor frequency of 13.81 MHz. The binary mixture exhibits the following phase sequence: isotropic—nematic—smectic A—reentrant nematic—crystal. The DMR spectrum of this sample is relatively well-resolved and permits the measurement of the Zeeman (T 1Z) and quadrupole (T 1Q) spin-lattice relaxation times for several distinct deuterons in 60CB-d 21. The present study considers only the ring deuterons. In fact, there are two sets of ring deuterons and we restrict our study to the set with the smaller quadrupolar splitting to avoid spectral overlap from the methyl deuterons. These relaxation times were simultaneously measured by the phase-cycled Jeener-Broekaert sequence, and the spectral densities Jp (ρω) (p = 1,2) were found to increase continuously with decreasing temperature. Molecular reorientation seems to be a dominant relaxation mechanism for the ring deuterons and is discussed in terms of a small step rotational diffusion model.

Vibrational and rotational relaxation of CDCl3 in nonpolar solvents

Vibrational and reorientational correlation functions and correlation times for the ν1 band of CDCl3 in the pure liquid and in solutions in carbon tetrachloride, n-hexane, and carbon disulfide have been obtained using spontaneous Raman scattering, and reorientational correlation times have been obtained from deuterium spin relaxation measurements. The concentration dependence of the reorientational correlation times in the CDCl3/CS2 and CDCl3/n-hexane solutions closely paralleled the solution viscosity, while in CDCl3/CCl4 mixtures, the correlation time exhibited little concentration dependence. These features reflect differences in the anisotropy of the intermolecular potential experienced by the CDCl3 molecules in these solutions. The reorientational correlation functions for the pure liquid exhibited time dependences different from that of the extended rotational diffusion models and a stochastic second-order memory function model. The temperature dependence of the vibrational relaxation times and vibrational second moments of ν1 in CDCl3 liquid is not consistent with Kubo-type dephasing models. The vibrational relaxation data indicate that collisional dephasing (Fischer–Lauberau model) is the probable mechanism of relaxation, but do not rule out contributions from energy relaxation via independent binary collisions. In CDCl3/CS2 mixtures, the band frequency and the bandwidth exhibit concentration dependence in accord with microscopic concentration fluctuations in the solution.