Modeling of proton spin relaxation in muscle tissue using nuclear magnetic resonance spin grouping and exchange analysis.

NMR spin relaxation experiments performed on healthy mouse muscle tissue at 40 MHz and 293 K are reported. The spin-lattice relaxation experiments were performed using different combinations of selective and nonselective radio frequency pulses. Relaxation experiments in the rotating frame at H1 = 10, 5 and 1 G are also reported. The experimental results were analyzed using the spin-grouping method, which yields the sizes of the resolved magnetization components as well as their T2's and T1's (or T1p's) for the nonexponential relaxation functions. These results were analyzed further for the exchange between different spin groups. It has been found that to explain all of these experimental data it was necessary to use a four-compartment model of the muscle tissue that consists of a lipid spin group, a "solid-like" spin group (mainly proteins), a "bulk water" spin group and a "bound water" spin group. The chemical exchange rate between "bulk" and "bound" water was found to be 29 +/- 9s-1 at room temperature. The exchange rate between the bound water and the solid moderator was estimated to be approximately 500 s-1.     

Extended Flip-back Schemes for Sensitivity Enhancement in Multidimensional HSQC-type Out-and-back Experiments

In many NMR experiments, only polarisation of a limited sub-set of all protons is converted into observable coherence. As recently shown by the “longitudinal” TROSY implementation (Pervushin et al. (2002) J. Am. Chem. Soc., 124, 12898–12902) and SOFAST-HMQC (Schanda and Brutscher (2005) J. Am. Chem. Soc., 127, 8014–8015), recovery of unused polarisation can be used indirectly and unspecifically to cool the proton lattice and, thus, accelerate re-equilibration for the selected proton subset. Here we illustrate transfer of this principle to HSQC-based multi-dimensional out-and-back experiments that exploit only polarisation of ¹⁵N-bound protons. The presented modifications to the pulse sequences can be implemented broadly and easily, extending standard flip-back of water polarisation to a much larger pool of protons that may comprise all non−¹⁵N-bound protons. The underlying orthogonal separation of H^N polarisation (selected by the main transfer path) from unused H^u polarisation (flipped-back on the recovery path) is thereby achieved through positive or negative selection by J-coupling, or using band-selective pulses. In practice, H^u polarisation recovery degrades mostly through cumulative pulse imperfections and transverse relaxation; we present, however, strategies to substantially minimise such losses particularly during interim proton decoupling. Depending on the protein’s relaxation properties and the extended flip-back scheme employed, we recovered up to 60% H^u equilibrium polarisation. The concomitant cooling of the proton lattice afforded substantial gains of more than 40%, relative to the water-only flip-back version, in the fast pulsing regime with re-equilibration delays τ much shorter than optimal (τ_opt = 1.25 · T₁(H^N)). These would be typically employed if resolution requirements dominate the total measurement time. Contrarily, if sensitivity is limiting and optimal interscan delays τ_opt can be set (optimal pulsing regime), the best of the presented flip-back schemes may still afford up to ca. 10% absolute sensitivity enhancement.     

Perspectives for sensitivity enhancement in proton-detected solid-state NMR of highly deuterated proteins by preserving water magnetization

In this work, we show how the water flip-back approach that is widely employed in solution-state NMR can be adapted to proton-detected MAS solid-state NMR of highly deuterated proteins. The scheme allows to enhance the sensitivity of the experiment by decreasing the recovery time of the proton longitudinal magnetization. The method relies on polarization transfer from non-saturated water to the protein during the inter-scan delay.     

Probing water accessibility in HET-s(218-289) amyloid fibrils by solid-state NMR

Despite the importance of protein fibrils in the context of conformational diseases, information on their structure is still sparse. Hydrogen/deuterium exchange measurements of backbone amide protons allow the identification hydrogen-bonding patterns and reveal pertinent information on the amyloid β-sheet architecture. However, they provide only little information on the identity of residues exposed to solvent or buried inside the fibril core. NMR spectroscopy is a potent method for identifying solvent-accessible residues in proteins via observation of polarization transfer between chemically exchanging side-chain protons and water protons. We show here that the combined use of highly deuterated samples and fast magic-angle spinning greatly attenuates unwanted spin diffusion and allows identification of polarization exchange with the solvent in a site-specific manner. We apply this measurement protocol to HET-s(218-289) prion fibrils under different conditions (including physiological pH, where protofibrils assemble together into thicker fibrils) and demonstrate that each protofibril of HET-s(218-289), is surrounded by water, thus excluding the existence of extended dry interfibril contacts. We also show that exchangeable side-chain protons inside the hydrophobic core of HET-s(218-289) do not exchange over time intervals of weeks to months. The experiments proposed in this study can provide insight into the detailed structural features of amyloid fibrils in general.     

Use of a water flip-back pulse in the homonuclear NOESY experiment

Summary A simple modification to the WATERGATE water suppression scheme [Piotto, M., Saudek, V. and Sklená, V. (1992) J. Biomol. NMR, 2, 661–665] is proposed. Radiation damping is used as an active element during the mixing time of a NOESY experiment, in order to obtain a reproducable state of the water magnetization at the end of the mixing time. Through the use of a water flip-back pulse and a gradient-tailored excitation scheme, we obtain both an excellent water suppression and a water magnetization close to equilibrium at the beginning of the acquisition time. We show experimentally that this modification results in a 20% gain in intensity for all signals when using a relaxation delay of 1.5 s, and also that avoiding a semisaturated state for the water magnetization allows the amide protons as well as other proton resonances to relax to equilibrium with their proper relaxation time.     

Some NMR Experiments and a Structure Determination Employing a {15N,2H} Enriched Protein

We present the results of studies of an aqueous sample of a highly {15N,2H} enriched protein, the SH3 domain from Fyn. Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7Å apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.     

Hydration and packing along the folding pathway of SH3 domains by pressure-dependent NMR

The volumetric properties associated with protein folding transitions reflect changes in protein packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation techniques are employed to probe the folding-unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathway can be measured. The two domains fold with rates that differ by approximately 3 orders of magnitude, so their folding dynamics must be probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial molar volumes can be obtained using low pressures (1-120 bar), thus minimizing perturbations to any of the states along the folding reaction coordinate. The volumetric data that were obtained are consistent with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that are at least partially hydrated. This work emphasizes the role of NMR spin relaxation in studying dynamic processes over a wide range of time scales.     

Detection of dynamic water molecules in a microcrystalline sample of the SH3 domain of α-spectrin by MAS solid-state NMR

Water molecules are a major determinant of protein stability and are important for understanding protein–protein interactions. We present two experiments which allow to measure first the effective T₂ decay rate of individual amide proton, and second the magnetization build-up rates for a selective transfer from H₂O to H^N using spin diffusion as a mixing element. The experiments are demonstrated for a uniformly ²H, ¹⁵N labeled sample of a microcrystalline SH3 domain in which exchangeable deuterons were back-substituted with protons. In order to evaluate the NMR experimental data, as X-ray structure of the protein was determined using the same crystallization protocol as for the solid-state NMR sample. The NMR experimental data are correlated with the dipolar couplings calculated from H₂O–H^N distances which were extracted from the X-ray structure of the protein. We find that the H^N T₂ decay rates and H₂O–H^N build-up rates are sensitive to distance and dynamics of the detected water molecules with respect to the protein. We show that qualitative information about localization and dynamics of internal water molecules can be obtained in the solid-state by interpretation of the spin dynamics of a reporter amide proton.     

NMR spin grouping and correlation exchange analysis. Application to low hydration NaDNA paracrystals.

The NMR spin-grouping technique is applied to low hydration oriented fibers of NaDNA to study the role of exchange in determining the apparent (observed) spin relaxation of the system. The analysis proceeds in three steps: first, the apparent proton relaxation is measured at high fields, with both selective and nonselective inversion pulse sequences, and in the rotating frame. The spin-grouping technique is used in all spin-lattice relaxation measurements to provide the optimum apparent relaxation characterization of the sample. Next, all apparent results are analyzed for exchange. In this analysis the results from the high field and rotating frame experiments (which probe the exchange at two different time scales) are correlated to determine the inherent (or true) spin relaxation parameters of each of the proton groups in the system. The results of selective inversion T1 measurements are also incorporated into the exchange analysis. Finally, the dynamics of each spin group are inferred from the inherent relaxation characterization. The low hydration NaDNA structure is such that the exchange between the protons on the water and those on the NaDNA is limited, a priori, to dipolar mixing. The results of the exchange analysis indicate that the dipolar mixing between water and NaDNA protons is faster than the spin diffusion within the NaDNA proton group itself. The spin-diffusion on the macromolecule is the bottleneck for the exchange between the water protons and the NaDNA protons. The water protons serve as the relaxation sink both at high fields and in the rotating frame for the total NaDNA-water spin bath. The inherent relaxation of the water is characteristic of water undergoing anisotropic motion with a fast reorientational correlation time about one axis (5 X 10(-10) less than or equal to tau r less than or equal to 8 X 10(-9)S) which is about three orders of magnitude slower than that of water in the bulk; and a slow tumbling correlation time for this axis (1.5 x 10(-7) less than or equal to tau t less than or equal to 8 x 10(-7)S) which is two orders of magnitude slower yet.     

Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

The identification of compounds that bind to a protein of interest is of central importance in contemporary drug research. For screening of compound libraries, NMR techniques are widely used, in particular the Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY) experiment. Here we present an optimized experiment, the polarization optimized WaterLOGSY (PO-WaterLOGSY). Based on a water flip-back strategy in conjunction with model calculations and numerical simulations, the PO-WaterLOGSY is optimized for water polarization recovery. Compared to a standard setup with the conventional WaterLOGSY, time consuming relaxation delays have been considerably shortened and can even be omitted through this approach. Furthermore, the robustness of the pulse sequence in an industrial setup was increased by the use of hard pulse trains for selective water excitation and water suppression. The PO-WaterLOGSY thus yields increased time efficiency by factor of 3–5 when compared with previously published schemes. These time savings have a substantial impact in drug discovery, since significantly larger compound libraries can be tested in screening campaigns. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of different water pools in solid-state NMR protein samples

We observed and characterized two distinct signals originating from different pools of water protons in solid-state NMR protein samples, namely from crystal water which exchanges polarization with the protein (on the NMR timescale) and is located in the protein-rich fraction at the periphery of the magic-angle spinning (MAS) sample container, and supernatant water located close to the axis of the sample container. The polarization transfer between the water and the protein can be probed by two-dimensional exchange spectroscopy, and we show that the supernatant water does not interact with protein on the timescale of the experiments. The two water pools have different spectroscopic properties, including resonance frequency, longitudinal, transverse and rotating frame relaxation times. The supernatant water can be removed almost completely physically or can be frozen selectively. Both measures lead to an enhancement of the quality factor of the probe circuit, accompanied by an improvement of the experimental signal/noise, and greatly simplify solvent-suppression by substantially reducing the water signal. We also present a tool, which allows filling solid-state NMR sample containers in a more efficient manner, greatly reducing the amount of supernatant water and maximizing signal/noise.     

QSim, a program for NMR simulations

We present QSim, a program for simulation of NMR experiments. Pulse sequences are implemented and analyzed in QSim using a mouse driven interface. QSim can handle almost any modern NMR experiment, using multiple channels, shaped pulses, mixing, decoupling, phase-cycling and pulsed field gradients. Any number of spins with any spin quantum number can, in theory, be used in simulations. Relaxation is accounted for during all steps of pulse sequences and relaxation interference effects are supported. Chemical kinetics between any numbers of states can be simulated. Both classical and quantum mechanical calculations can be performed. The result of a simulation can be presented either as magnetization as a function of time or as a processed spectrum.     

Use of relaxation-edited one-dimensional and two dimensional nuclear magnetic resonance spectroscopy to improve detection of small metabolites in blood plasma

The 1H nuclear magnetic resonance (NMR) spectra of biological samples, such as blood plasma and tissues, are information rich but data complex owing to superposition of the resonances from a multitude of different chemical entities in multiple-phase compartments, hampering detection and subsequent resonance assignments. To overcome these problems, several spectral-editing NMR experiments are described here, combining spin-relaxation filters (based on T(1), T(rho), and T(2)) with both one-dimensional and two-dimensional (2D) NMR spectroscopy. These techniques enable the separation of NMR resonances based on their relaxation times and allow simplification of the complex spectra. In this paper, the approach is exemplified using a control human blood plasma, which is a complex mixture of proteins, lipoproteins, and small-molecule metabolites. In the case of T(1rho)- and T(2)-edited 2D NMR experiments, a "flip-back" pulse was introduced after the relaxation editing to make the phase cycling of the "relaxation filter" and the 2D NMR part independent, thus enabling easy implementation of the phase-sensitive 2D NMR experiments. These methods also permit much higher receiver gains to be used to reduce digitization error, in particular, for the small resonances, which are sometimes vitally important for metabonomics studies. Both pulse sequences and experimental results are discussed for T(1)-, T(1rho)-, and T(2)-filtered COSY, T(2)-filtered phase-sensitive DQF-COSY, and T(1), T(1rho)-, and T(2)-filtered TOCSY NMR.     

Proton NMR spin grouping and exchange in dentin.

The nuclear magnetic resonance spin-grouping technique has been applied to dentin from human donors of different ages. The apparent T2, T1, and T1 rho have been determined for natural dentin, for dentin which has been dried in vacuum, and for dried dentin which has been rehydrated in an atmosphere with 75% relative humidity. All apparent spin relaxation has been analyzed for exchange between the spin groups in which the dentin protons exist; the analyses incorporate the results of selective inversion recovery T1 measurements which better probe the effects of exchange. The exchange analyses of the high fields and rotating frame spin-lattice relaxation have also been correlated to determine uniquely the inherent relaxation parameters of the proton spin groups constituting the dentin magnetization. The natural dentin contains protons on water, protein, and hydroxy apatite; these spins contribute 50%, 45%, and 5% to the total dentin proton magnetization, respectively. The water exists in three distinct environments, the dynamics of each environment has been modeled. In the natural dentin 30% of the water undergoes uni-axial reorientation. 52% of the water has similar relaxation characteristics to bound water hydrating a large molecule, and the majority of the remaining water acts as bulk water undergoing isotropic reorientation. The results are independent of the age of the donor.     

How does it really move? Recent progress in the investigation of protein nanosecond dynamics by NMR and simulation

Nuclear magnetic resonance (NMR) spin relaxation experiments currently probe molecular motions on timescales from picoseconds to nanoseconds. The detailed interpretation of these motions in atomic detail benefits from complementarity with the results from molecular dynamics (MD) simulations. In this mini-review, we describe the recent developments in experimental techniques to study the backbone dynamics from ¹⁵N relaxation and side-chain dynamics from ¹³C relaxation, discuss the different analysis approaches from model-free to dynamics detectors, and highlight the many ways that NMR relaxation experiments and MD simulations can be used together to improve the interpretation and gain insights into protein dynamics.     

Formation of microsomal cytochrome P-450 complexes studied by the NMR relaxation of water

Cytochrome P-450 in microsomes from liver of phenobarbital treated and control rats has been studied by light absorption and by magnetic resonance methods (EPR and NMR). The nuclear relaxation rate of water protons was measured for microsomal suspensions in the presence of various reactants of Type I and II. The change of relaxation rates correlates well with the spin state conversion of the heme iron. No competition between eventual inner-sphere water molecules and the reactants seems to occur. The temperature dependence of the low spin to high spin equilibrium was studied by light absorption and was accounted for in the temperature variation of the molar relaxation rates of the two spin states.     

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.     

Direct estimation of base-pair exchange kinetics in oligo-DNA by a combination of NOESY and ROESY experiments.

A new method for the determination of the kinetics of exchange of the imino protons of DNA duplex is reported using a combination NOESY and ROESY experiments at short mixing times (< or = 20 ms). These results have been compared with the commonly used longitudinal relaxation approach through the T1 measurement. To calculate kex and pi ex by ROESY-NOESY experiment, the volume of the cross-peaks between imino protons and water in the NOESY and ROESY spectra have been measured separately from the magnetization term. This work shows that the present approach for the measurement of the kinetics of slow exchanging imino protons of DNA duplex is comparable to the saturation recovery experiment in which the exchange rate can be accelerated by the addition of a base catalyst. The present ROESY-NOESY approach has been found to be particularly useful and reasonably accurate for the measurement of exchange kinetics of both the fast- and slow-exchanging imino protons in DNA duplex both under non-physiological and physiological condition where the saturation recovery method can not be used.     

1H-NMR relaxation studies of native and thermally denatured lysozyme solutions: an isotope dilution experiment

1H-NMR relaxation times are reported for native and thermally denatured lysozyme aqueous solutions measured as the function of the proton mole fraction in the sample. A two-exponential character of proton longitudinal relaxation function was observed for native lysozyme solutions: the fast component was attributed to the non-exchangeable protein protons, the slow one to water protons. Purely exponential decay of longitudinal magnetization was observed for the thermally denatured samples. This has been explained in terms of a fast spin exchange model. The contributions of the protein protons to the water proton relaxation rate in native and thermally denatured samples were determined, too.