Magnetic Relaxation Dispersion Experiments Research Articles

Fast Microsecond Dynamics of the Protein-Water Network in the Active Site of Human Carbonic Anhydrase II Studied by Solid-State NMR Spectroscopy.

Protein-water interactions have widespread effects on protein structure and dynamics. As such, the function of many biomacromolecules can be directly related to the presence and exchange of water molecules. While the presence of structural water sites can be easily detected by X-ray crystallography, the dynamics within functional water-protein network architectures is largely elusive. Here we use solid-state NMR relaxation dispersion measurements with a focus on those active-site residues in the enzyme human carbonic anhydrase II (hCAII) that constitute the evolutionarily conserved water pocket, key for CAs' enzymatic catalysis. Together with chemical shifts, peak broadening, and results of molecular dynamics (MD) and DFT shift calculations, the relaxation dispersion data suggest the presence of a widespread fast μs-time-scale dynamics in the pocket throughout the protein-water network. This process is abrogated in the presence of an inhibitor which partially disrupts the network. The time scale of the protein-water pocket motion coincides both with the estimated residence time of Zn-bound water/OH- in the pocket showing the longest lifetimes in earlier magnetic relaxation dispersion experiments as well as with the rate-limiting step of catalytic turnover. As such, the reorganization of the water pocket:enzyme architecture might constitute an element of importance for enzymatic activity of this and possibly other proteins.

Transient Access to the Protein Interior: Simulation versus NMR

Many proteins rely on rare structural fluctuations for their function, whereby solvent and other small molecules gain transient access to internal cavities. In magnetic relaxation dispersion (MRD) experiments, water molecules buried in such cavities are used as intrinsic probes of the intermittent protein motions that govern their exchange with external solvent. While this has allowed a detailed characterization of exchange kinetics for several proteins, little is known about the exchange mechanism. Here, we use a millisecond all-atom MD trajectory produced by Shaw et al. (Science2010, 330, 341) to characterize water exchange from the four internal hydration sites in the protein bovine pancreatic trypsin inhibitor. Using a recently developed stochastic point process approach, we compute the survival correlation function probed by MRD experiments as well as other quantities designed to validate the exchange-mediated orientational randomization (EMOR) model used to interpret the MRD data. The EMOR model is found to be quantitatively accurate, and the simulation reproduces the experimental mean survival times for all four sites with activation energy discrepancies in the range 0-3 kBT. On the other hand, the simulated hydration sites are somewhat too flexible, and the water flip barrier is underestimated by up to 6 kBT. The simulation reveals that water molecules gain access to the internal sites by a transient aqueduct mechanism, migrating as single-file water chains through transient (<5 ns) tunnels or pores. The present study illustrates the power of state-of-the-art molecular dynamics simulations in validating and extending experimental results.

Impact of wettability on moisture transport at mesoscale in porous materials

A multiscale NMR method is used for probing the proton dynamics in control pore glasses (CPG) standing in the meso and macro scale ranges. The porous material has been synthetized in our laboratory following the sol–gel route as well as spinodal decomposition. Special heat treatment has modified the proton species at the pore surface. 1H high field NMR spectroscopy has been realized under controlled relative humidity conditions. This has allowed a clear identification of the different proton species present during the kinetics of hydration from RH=0% up to 98%. We focused on the nuclear magnetic relaxation dispersion experiments to probe specifically the proton dynamics in CPG pores with and without methyl groups at the surface. A detailed analysis of the frequency dependences of the longitudinal relaxation rates in these two materials evidences the impact of microscopic wettability on moisture transport in meso- and macro-porous materials. The surface modification has allowed a frequency rescaling such that different regimes of relaxation dependence can be fully explored in a larger frequency range than that explored by FFC relaxometry. We believe that such an NMR study realized on model porous materials is of particular importance for studying the moisture transport between the meso- and macro-porous networks of cement-based materials.

Biomolecular hydration: from water dynamics to hydrodynamics.

Thermally driven rotational and translational diffusion of proteins and other biomolecules is governed by frictional coupling to their solvent environment. Prediction of this coupling from biomolecular structures is a longstanding biophysical problem, which cannot be solved without knowledge of water dynamics in an interfacial region comparable to the dry protein in volume. Efficient algorithms have been developed for solving the hydrodynamic equations of motion for atomic-resolution biomolecular models, but experimental diffusion coefficients can be reproduced only by postulating hundreds of rigidly bound water molecules. This static picture of biomolecular hydration is fundamentally inconsistent with magnetic relaxation dispersion experiments and molecular dynamics simulations, which both reveal a highly dynamic interface where rotation and exchange of nearly all water molecules are several orders of magnitude faster than biomolecular diffusion. Here, we resolve this paradox by means of a dynamic hydration model that explicitly links protein hydrodynamics to hydration dynamics. With the aid of this model, bona fide structure-based predictions of global biomolecular dynamics become possible, as demonstrated here for a set of 16 proteins for which accurate experimental rotational diffusion coefficients are available.

Dynamics of water and oil at the solid–liquid interface in macroporous media and reservoir rocks

Nuclear magnetic relaxation dispersion experiments at different temperatures, using the magnetic field-cycling method, are reported. These experiments allow the obtaining of original information about water or oil molecular dynamics at solid–liquid interface in porous media, such as grain packing or reservoir rocks. These results on molecular dynamics at the pore surface are of real interest for oil-recovery. The water surface diffusion coefficients are compared to the volume self-diffusion coefficients of water in pores, measured by PGSE method, the latter values being more than an order of magnitude higher than the surface ones. Nous avons obtenu des informations originales sur la dynamique de l’eau et de l’huile aux interfaces solide–liquide de milieux poreux plus ou moins complexes, comme les empilements granulaires et les roches réservoirs. Ces informations proviennent d’expériences de relaxation magnétique nucléaire du proton en champ magnétique cyclé à différentes températures. Ces résultats sur la dynamique des molécules à la surface des pores présentent un réel intérêt dans le domaine de la récupération du pétrole. Des mesures de diffusion par la méthode des gradients de champ pulsés permettent une comparaison entre les coefficients de diffusion en surface et en volume, ces derniers étant de plus d’un ordre de grandeur supérieurs à ceux en surface.

First Noncovalently Bound Calix[4]arene-GdIII-Albumin Complex

The gadolinium(III) complex of the new water-soluble calix[4]arene tetraamide 1 is the first calix[4]arene that noncovalently binds to human serum albumin (HSA), as confirmed by nuclear magnetic relaxation dispersion experiments. Thus, in principle, it should be suitable as a contrast agent for NMR imaging applications.

Anomalous surface diffusion of water compared to aprotic liquids in nanopores.

1H nuclear magnetic relaxation dispersion experiments show remarkable differences between water and acetone in contact with microporous glass surfaces containing trace paramagnetic impurities. Analyzed with surface relaxation theory on a model porous system, the data obtained for water show that proton surface diffusion limited by chemical exchange with the bulk phase permits long-range effectively one-dimensional exploration along the pores. This magnetic-field dependence coupled with the anomalous temperature dependence of the relaxation rates permits a direct interpretation in terms of the proton translational diffusion coefficient at the surface of the pores. A universal rescaling applied to these data collected for different pore sizes and on a large variety of frequencies and temperatures, supports this interpretation. The analysis demonstrates that acetone diffuses more slowly, which increases the apparent confinement and results in a two-dimensional model for the molecular dynamics close to surface relaxation sinks. Surface-enhanced water proton diffusion, however, permits the proton to explore a greater spatial extent of the pore, which results in an apparent one-dimensional model for the diffusive motions of the water that dominate nuclear spin relaxation.

Anomalous Surface Diffusion of Water in Nanopores

AbstractProton nuclear magnetic relaxation dispersion experiments show remarkable differences between water and aprotic liquids in contact with microporous glass surfaces containing trace paramagnetic impurities. Proton surface diffusion permits long range effectively 1-dimensional exploration along the pores for water. This gives a direct measurement of the translational diffusion coefficient at the pore surface.

Electron relaxation and solvent accessibility of the metal site in wild-type and mutated azurins as determined from nuclear magnetic relaxation dispersion experiments

The interaction of water molecules with copper in wild-type azurin and different site-directed mutants of the coordinated residues is studied by nuclear magnetic relaxation dispersion. Different degrees of solvent accessibility are found. The low relaxivity of wild-type azurin agrees with a solvent-protected copper site in solution, the closest water being found at a distance of more than 5 A from the copper. This low relaxivity contrasts with the relatively large relaxivity of the His46Gly and His117Gly azurin mutants, which shows clear evidence of copper-coordinated water. The data on the latter mutants are best analyzed in terms of one and two water molecules coordinated to the copper in His46Gly and His117Gly, respectively. The Met121His azurin mutant shows an intermediate behavior. The data are analyzed in terms of an increased solvent accessibility with respect to the wild-type azurin, resulting in semi-coordination of water at low pH. These different modes of coordination lead to different geometries, ranging from the trigonal type 1 site of wild-type azurin to the tetragonal type 2 copper sites of the His117Gly and His46Gly azurin mutants through a so-called type 1.5 site of the Met121His mutant. A correlation is found between the relaxation time (τs) of the unpaired electron of copper(II) and the geometry of the metal site: as the tetragonal character decreases the relaxation becomes significantly faster. τs values of ≤1 ns are found for the tetrahedrally distorted type 1 and type 1.5 sites and of 5–15 ns for the tetragonal type 2 sites.