D2O Environment Research Articles

Water Networks and Allosteric Scaffolds ‐ What Does Water Have to do with Protein Dynamics?

The thermophilic enzyme Fluoroacetate Dehalogenase (FAcD) has been well characterized by crystallography and NMR. FAcD (RPA1163) is a homodimer alpha/beta hydrolase that breaks down haloacetates (including fluoroacetate, iodoacetate, and chloroacetate) into glycolate and a halogen ion. FAcD achieves this by half‐of‐sites activity, where the homodimer adopts a rare asymmetric state that is primed to capture substrate which must then diffuse along an 11 Å cavity before engaging the active site and adopting the Michaelis intermediate. This triggers release of ~30 bound water molecules, thereby entropically favouring the forward reaction. FAcD is thus an ideal model system to pursue paradigm shifting questions regarding allostery, interprotomer communication, and the role of hydrogen bonded water networks in catalysis ‐ all from an ensemble perspective. Here we examine how deuterium water (D2O) changes the dynamic and allosteric mechanisms of this system. Compared to water, D2O results in an increase in viscosity and has stronger hydrogen bonds. Through 19F NMR kinetic experiments we determine that despite the higher viscosity enzyme activity is increased in the D2O environment. Additionally, 19F NMR experiments reveal increased dynamics in D2O. This provides key insight into discrete hydrogen‐bonded water networks that are key to stabilization of allosteric processes associated with the reaction coordinate.Support or Funding InformationNatural Sciences and Engineering Research Council (NSERC)

Longer-Lasting Electron-Based Microscopy of Single Molecules in Aqueous Medium.

Use of electron-based microscopy in aqueous media has been held back because aqueous samples tend to suffer from water radiolysis and other chemical degradation caused by the high energy of incident electrons. Here we show that aqueous liquid pockets in graphene liquid cells at room temperature display significantly improved stability when using deuterated water, D2O. Reporting transmission electron microscopy (TEM) experiments based on common imaging conditions, we conclude that use of D2O outperforms adding radical scavengers to H2O regardless of imaging details; it increases the lifetime of dissolved organic macromolecules by a factor of 2-5, and it delays by even longer the appearance of radiolysis-induced bubbles, by a factor of time up to 10. We quantify statistically the consequences of minimizing the electron voltage and dose and conclude that the D2O environment increases sample longevity without noticeable sacrifice of contrast that is critical for direct imaging of weakly scattering organic macromolecules and biomolecules.

The Effect of D2O on the Inactivation Kinetics and Recovery from Slow Inactivation of Shaker-IR K+ Channels

In the absence of N-type inactivation Shaker potassium channels display slow (C-type) inactivation which is stabilized by a multipoint hydrogen-bond network behind at the selectivity filter in eukaryotic KV channels. The selectivity filter is sterically locked in the inactive conformation by buried water molecules which prevent recovery from inactivation. To further test the critical role of these, structural water” molecules in the conformational stability of the selectivity filter we studied the effects of substituting water for heavy water (deuterium oxide, D2O in the solutions) on various gating transitions in Shaker-IR channels having the following mutations and allowing slow inactivation at various rates: T449A, T449A/I470A and T449K/I470C. All channels were transiently expressed in tsA_201 cells and ionic currents were recorded in excised inside-out patches. 2.0-s-long depolarizing pulses from a holding potential of −120 mV to +50 mV were applied to measure the current through the channels. The extracellular application of D2O dramatically slowed entry into the inactivated state, the inactivation time constats were τi,D2O = 189±48 ms (n=5) (T449A, τi,H2O = 72±13 (n=5)), τi,D2O = 418±63 ms (n=4) (T449A/I470A, τi,H2O = 247±90 (n=5)), τi,D2O = 60±21 ms (n=6) (T449K/I470C, τi,H2O = 31±15 ms (n=10)), respectively. In contrast, applying D2O from the intracellular side did not change the inactivation kinetics. The kinetics of recovery from slow inactivation was also slowed down in the D2O environment. Our macroscopic current measurements confirm that water molecules may have access to the region behind the selctivity filter only from the external solution and the binding of these ‘hidden’ molecules slows the development of inactivation and that of the recovery process.

Car-Parrinello Molecular Dynamics Study for the Isotope Effect on OH Vibration in Ice Ih

The stretching vibration of OH of ice Ih is studied by Car-Parrinello molecular dynamics in regarding the effect of mixed H/D contamination while the vibrational spectrum is considered by velocity-velocity autocorrelations of the sampled ensemble. When hydrogen atoms are immersed randomly into the deuterated ice, a typical vibrational frequency of OH stretching mode is observed to be similar to that from the pure H2O ice. When focusing on the correlation of isolated neighboring OH stretching, a narrower and blue shifted peak is observed at the high frequency range as a result of the screening from the complex many body correlations by D2O environment. It is also specifically related to the symmetric intermolecular correlations between neighboring OH stretching modes. More enhanced high frequency range can be explained by the expansion of such two body correlations to collective many body correlations among all possible OH stretching modes. This contribution becomes important when it involves in chemical interactions via excitation of such vibrational states.

Silk Layering As Studied with Neutron Reflectivity

Neutron reflectivity (NR) measurements of ultrathin surface films (below 30 nm) composed of Bombyx mori silk fibroin protein in combination with atomic force microscopy and ellipsometry were used to reveal the internal structural organization in both dry and swollen states. Reconstituted aqueous silk solution deposited on a silicon substrate using the spin-assisted layer-by-layer (SA-LbL) technique resulted in a monolayer silk film composed of random nanofibrils with constant scattering length density (SLD). However, a vertically segregated ordering with two different regions has been observed in dry, thicker, seven-layer SA-LbL silk films. The vertical segregation of silk multilayer films indicates the presence of a different secondary structure of silk in direct contact with the silicon oxide surface (first 6 nm). The layered structure can be attributed to interfacial β-sheet crystallization and the formation of well-developed nanofibrillar nanoporous morphology for the initially deposited silk surface layers with the preservation of less dense, random coil secondary structure for the layers that follow. This segregated structure of solid silk films defines their complex nonuniform behavior in the D(2)O environment with thicker silk films undergoing delamination during swelling. For a silk monolayer with an initial thickness of 6 nm, we observed the increase in the effective thickness by 60% combined with surprising decrease in density. Considering the nanoporous morphology of the hydrophobic silk layer, we suggested that the apparent increase in its thickness in liquid environment is caused by the air nanobubble trapping phenomenon at the liquid-solid interface.

Two-Step Desorption Process of Au Nanoparticles in D2O Suspension on Amino-Terminated SiO2/Si Substrate Induced by Small Thiol Molecules

We investigated the desorption process of Au nanoparticles immobilized on an amino-terminated SiO2/Si surface in a D2O environment by in situ attenuated total reflection surface enhanced infrared absorption spectroscopy. The advantages of IR spectroscopy over other methods were demonstrated; the IR spectra are not only sensitive to the changing particle density at the surface, but also to chemical environmental changes. Therefore, the gained information obtained by IR spectroscopy and scanning electron microscopy allows us to propose a microscopic model for the desorption process of the Au nanoparticles from the substrate.

Induction of direct and indirect single-strand breaks in human cell DNA by far- and near-ultraviolet radiations: action spectrum and mechanisms.

Abstract— An action spectrum for the immediate induction in DNA of single‐strand breaks (SSBs, frank breaks plus alkali‐labile sites) in human P3 teratoma cells in culture by monochromatic 254‐, 270‐, 290‐, 313‐, 334‐, 365‐, and 405‐nm radiation is described. The cells were held at +0.5d̀C during irradiation and were Iysed immediately for alkaline sedimentation analysis following the irradiation treatments. Linear fluence responses were observed over the fluence ranges studied for all energies. Irradiation of the cells in a D2O environment (compared with the normal H2O environment) did not alter the rate of induction of SSBs by 290‐nm radiation, whereas the D2O environment enhanced the induction of SSBs by 365‐ and 405‐nm irradiation. Analysis of the relative efficiencies for the induction of SSBs, corrected for quantum efficiency and cellular shielding, revealed a spectrum that coincided closely with nucleic acid absorption below 313 nm. At longer wavelengths, the plot of relative efficiency vs. wavelength contained a minor shoulder in the same wavelength region as that observed in a previously obtained action spectrum for stationary phase Bacillus subtilis cells. Far‐UV radiation induced few breaks relative to pyrimidine dimers, whereas in the near‐UV region of radiation, SSBs account for a significant proportion of the lesions relative to dimers, with a maximum number of SSBs per lethal event occurring at 365‐nm radiation.

Moisture Distribution Measurements in Adhesive-Bonded Composites Using the D(3He,p)4He Reaction

Adhesive bonding of composite materials for many aircraft components offers a distinct advantage in weight and cost reduction compared to similar structures that have been joined by riveting. However, the long term performance of adhesive-bonded components depends on the degree and rate of moisture absorption by the adhesive in the service environment. To investigate the rate and the mechanism of water transport in adhesive-bonded composite materials, a nuclear reaction analysis method based on the D(3He,p)4He reaction is used to measure the moisture distributions. Samples of graphite/epoxy composite materials were bonded with an epoxy adhesive and isothermally conditioned in a controlled D2O environment at 70% relative humidity and 77°C for various exposure times. The moisture profiles were measured along the adhesive (adhesive scan) as well as through the thickness of the bonded joint (transverse scan). The dimensions of the probing beam were 125 ?m × 125 ?m for the adhesive scan and 25 ?m × 200 ?m for the transverse scan. Absolute deuterium concentrations were determined by comparison of the proton yield from the composite/adhesive to that from reference standards. Calculations from diffusion models of water transport based on parameters determined from bulk measurement techniques are compared to the measured profile and the agreement indicates that classical Fickian diffusion describes the transport of moisture in these materials.

Stability characteristics of deuterated myoglobin.

AbstractPurified sperm whale myoglobin was deuterated by being exposed to pD 3.5 in D2O buffer for 1 hr, then raised to pD 10.6 for an additional hour, and finally brought to neutrality in a D2O environment. Control myoglobin was similarly treated in H2O. The specific rotation at 233 mμ and/or the absorbance in the Soret region were used to follow the helix‐coil transition of myoglobin subjected to denaturation by acid, alkali, urea and guanidine. Deuterated and control myoglobin had similar 50% transition points in the four denaturing media studied (acid: pH 4.4, pD 4.9; alkali: pH 9.4, pD 10.0; urea, 7.2M; guanidine, 2.5M). The shift toward the alkaline side of 0.5 or 0.6 units of the transition induced by either acid or alkaline denaturation reflects only the weakened acidity of ionizable groups in deuterium systems. Deuterated myoglobin in 3.25M guanidine had a 20% faster denaturation rate than that of control. Acid, urea, and guanidine denaturation curves showed fairly steep transitions, taken as indicative of a one‐step process involving only two states (native and denatured molecules). Supporting this conclusion was the fact that plots of absorption and polarimetry measurements of the helix‐coil transition induced by either acid or guanidine could be superimposed.