Bulk D2O Research Articles

The partitioning of primary alcohols into the aggregates of gemini amphiphiles determined from diffusion NMR experiments.

The partition constants (p-values) of primary alcohols in solutions containing aggregates of symmetric gemini surfactants of the family N,N'-dimethyl, N-dialkyl-α,ω-alkanediammonium dibromide (m-s-m = symmetric gemini surfactants) have been computed from the measured values of their diffusion coefficients obtained from NMR-diffusion experiments. From the p-values, both mole-fraction and concentration-based partition coefficients and Gibbs energies of transfer for the alcohols from the bulk D2O phase to the gemini aggregate phase have been calculated. As expected, the Gibbs energy of transfer decreased linearly with an increase in the alcohol carbon length for each of the primary alcohol/gemini amphiphile series studied. The Gibbs transfer energy increment per CH2 for the alcohols was consistent for all the alcohol/gemini amphiphile series and was in excellent agreement with the values measured for the same primary alcohol series in conventional single-headed, single-tailed surfactants. Surprisingly, the partition coefficients of the alcohols in the symmetric gemini aggregates exhibited little, if any, dependence on the spacer length of the gemini amphiphiles and were remarkably consistent as the length of the main surfactant chain increased at constant spacer length. When these results are compared to the partition coefficients of the same alcohols in corresponding monomeric surfactants, we observe little difference in the thermodynamic driving forces governing the transfer of alcohols from water to the aggregates of either monomeric or symmetric gemini surfactants.

Ionic liquid-based reverse micelles. Use of hydrogen–deuterium exchange as a tool for surfactant self-assembly characterization

We report the study of the rate for hydrogen–deuterium (H-D) exchange in the cation of the ionic liquid-surfactant 1-butyl-3-methylimidazolium 1,4-bis-2-ethylhexylsulfosuccinate (bmim-AOT) when it forms reverse micelles (RMs). The results were compared with those found for bmim-AOT dissolved in D2O showing an inhibition larger than 500-fold on going from bulk D2O to the reverse micelle. Also, the effect of varying water content and surfactant concentration of the RMs on the deuterium exchange was evaluated by 1H NMR technique. The results demonstrate that the H-D exchange at the C(2) position of bmim+ when it forms RMs significantly reflects the properties of the micellar microenvironment, specifically of the micellar interface due to the localization of bmim+ in the interfacial region, intercalated between the AOT moiety. The rate of deuterium exchange is directly related to the properties of interfacial water, which, in turn, is closely related to the water-surfactant interaction. Thus, the analysis of deuterium exchange in the 2-position of bmim+ is proposed as a useful tool to explore the properties of RMs formed by this cation, with the advantage of being a non-invasive technique since the ionic liquid-surfactant itself acts as the molecular probe.

Interaction of Water with Graphene/Ir(111) Studied by Vibrational Spectroscopy.

Water in confinement exhibits altered properties in molecular arrangement, bonding, and interaction with its neighboring environment, as compared to its bulk counterpart. In this work, periodically arranged D2O nano droplets of ∼1 nm size on top of a graphene/iridium moiré superstructure were investigated by Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS) under ultrahigh vacuum conditions at ∼120 K. The IR bands of D2O clusters differ significantly from those observed for bulk D2O amorphous solid water or crystalline ice phases. Blue-shifted symmetric and asymmetric stretching bands with narrower band widths and modified band intensity ratios were observed, pointing to an enhanced internal order and a reduced nearest neighbor distance. Furthermore, two IR bands of "dangling" deuterium atoms were detected originating from threefold coordinated water molecules at the surface of the clusters and at their interface to the graphene layer. The latter arose only with the transition from the water clusters to an amorphous solid water layer. We propose that upon coalescence, opposing local dipoles trigger a hydrogen bond rearrangement at the interface. Our results represent a first step toward an atomistic understanding of water in confinement.

Improving yields of deuterated, methyl labeled protein by growing in H2O.

Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the 15N-1H TROSY and 13C-1H3 methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E. coli) during growth on media prepared in D2O leads to many difficulties and limitations, such as cell toxicity, decreased yield, and the need to unfold or destabilize proteins for back exchange of amide protons. These issues are exacerbated for non-ideal systems such as membrane proteins. Expression of protein during growth in H2O, with the addition of 2H-labeled amino acids derived from algal extract, can potentially avoid these issues. We demonstrate a novel fermentation methodology for high-density bacterial growth in H2O M9 medium that allows for appropriate isotopic labeling and deuteration. Yields are significantly higher than those achieved in D2O M9 for a variety of protein targets while still achieving 75-80% deuteration. Because the procedure does not require bulk D2O or deuterated glucose, the cost per liter of growth medium is significantly decreased; taking into account improvements in yield, these savings can be quite dramatic. Triple-labeled protein is also efficiently produced including specific 13CH3 labeling of isoleucine, leucine, and valine using the traditional ILV precursors in combination with an ILV-depleted mix of 2H/15N amino acids. These results are demonstrated for the membrane protein sensory rhodopsin II and the soluble proteins human aldoketoreductase AKR1c3, human ubiquitin, and bacterial flavodoxin. Limitations of the approach in the context of very large molecular weight proteins are illustrated using the bacterial Lac repressor transcription factor.

Water-anion hydrogen bonding dynamics: Ultrafast IR experiments and simulations.

Many of water's remarkable properties arise from its tendency to form an intricate and robust hydrogen bond network. Understanding the dynamics that govern this network is fundamental to elucidating the behavior of pure water and water in biological and physical systems. In ultrafast nonlinear infrared experiments, the accessible time scales are limited by water's rapid vibrational relaxation (1.8 ps for dilute HOD in H2O), precluding interrogation of slow hydrogen bond evolution in non-bulk systems. Here, hydrogen bonding dynamics in bulk D2O were studied from the perspective of the much longer lived (36.2 ps) CN stretch mode of selenocyanate (SeCN-) using polarization selective pump-probe (PSPP) experiments, two-dimensional infrared (2D IR) vibrational echo spectroscopy, and molecular dynamics simulations. The simulations make use of the empirical frequency mapping approach, applied to SeCN- for the first time. The PSPP experiments and simulations show that the orientational correlation function decays via fast (2.0 ps) restricted angular diffusion (wobbling-in-a-cone) and complete orientational diffusive randomization (4.5 ps). Spectral diffusion, quantified in terms of the frequency-frequency correlation function, occurs on two time scales. The initial 0.6 ps time scale is attributed to small length and angle fluctuations of the hydrogen bonds between water and SeCN-. The second 1.4 ps measured time scale, identical to that for HOD in bulk D2O, reports on the collective reorganization of the water hydrogen bond network around the anion. The experiments and simulations provide details of the anion-water hydrogen bonding and demonstrate that SeCN- is a reliable vibrational probe of the ultrafast spectroscopy of water.

Structure of aqueous water films on textured -OH-terminated self-assembled monolayers.

We report the thickness and interfacial molecular structure of thin (1-3 nm) aqueous films supported on hydroxyl-terminated self-assembled monolayers over a silver substrate. The water film structure is studied as a function of varying the monolayer's methylene chain lengths. Analysis techniques include ellipsometry, contact angle, and polarization modulation reflection adsorption infrared spectroscopy. The aqueous film thicknesses follow 4-mercaptobutanol (4-MBU) > 11-mercaptoundecanol (11-MUD) > 6-mercaptohexanol (6-MHE) > 9-mercaptononanol (9-MNO). Water contact angle measurements across the same surfaces are very similar; however, vibrational spectroscopic analysis of the films shows that intermolecular bonding patterns of D2O are significantly different from those of bulk D2O. This evokes unique interfacial molecular architectures for each of these films. The structural differences depend on the nature of the SAM structure and resulting water-SAM interactions, which are evident from PM-IRRAS data. Spectroscopic peak intensity ratios of ν(O-D) modes suggest more asymmetric hydrogen-bonded D2O character near 9-MNO surfaces, whereas 4-MDU, 6-MHE, and 11-MUD surfaces exhibit increasingly symmetric hydrogen-bonded D2O character. From this, we propose a model for film structure.

Neutron reflectometry characterization of PEI–PSS polyelectrolyte multilayers for cell culture

Using Neutron Reflectometry (NR), polyelectrolyte multilayer (PEM) films made by layer-by-layer (LbL) deposition of a strong polycation (polyethylene imine [PEI]) and a polyanion (polystyrene sulfonate [PSS]) have been characterized. PEI terminated samples with a total of 5, 7, and 9 layers were deposited on a quartz substrate and studied under three different environmental conditions (i.e., dry air, 100% relative humidity, and bulk water). We were able to model all the measurements at three different contrast conditions using one simple, physically reasonable and consistent model, which led to a firm understanding of the structure of the systems. The PEM thickness was found to vary linearly with the number of layers deposited. Thin film structures formed using the LbL method were constituted of two distinctive regions, i.e., the bottom and top strata. When measured in dry air and D2O vapors, the ∼30 to 50 A thick bottom stratum was found to consist of loosely packed polymers (i.e. 30% polymer by volume). This region could have resulted from an island type of deposition during the initial stages of LbL assembly. In contrast, the thickness of the top strata, which consisted of densely packed polymers (i.e. 100% polymer by volume when measured in dry air), was found to vary linearly with the number of layers deposited. Upon exposure to D2O saturated vapors, it was observed that the top and bottom strata absorbed significant quantities of heavy water, accompanied with PEM swelling. We estimated that in this case, the top strata comprise ca. 57% (v/v) polymer and 43% (v/v) D2O for 7- and 9-layered samples. No further swelling of the PEM samples was observed when they were exposed to bulk D2O. Nevertheless, the entire polymeric system underwent a rearrangement leading towards the homogenization of the multilayered structure, suggested by the decreased scattering contrast between the top and bottom strata. We also performed studies to assess the cytocompatibility of 7-layered PEM structures. Two different cell types, fibroblasts (3T3) and human embryo kidney cells (HEK-293), were seeded on the polyelectrolyte multilayer, and the cell coverage was monitored by optical microscopy at varying times. Our observations confirmed that cells adhered and spread on PEM substrates, which showed no sign of immediate toxicity. Therefore, such multilayers proved to be a suitable support for 3T3 and HEK-293 cell growth.

Ultrafast intermolecular energy transfer in heavy water

We report on a study of the vibrational energy relaxation and resonant vibrational (Förster) energy transfer of the OD vibrations of D2O and mixtures of D2O and H2O using femtosecond mid-infrared spectroscopy. We observe the lifetime of the OD vibrations of bulk D2O to be 400 +/- 30 fs. The rate of the Förster energy transfer is measured via the dynamics of the anisotropy of the OD vibrational excitation. For a solution of 0.5% D2O in H2O, resonant energy transfer is negligible, and the anisotropy shows a single exponential decay with a time constant of 2.6 +/- 0.1 ps, representing the time scale of the molecular reorientation. With increasing concentration, the anisotropy decay becomes faster and non-exponential, showing the increased contribution of resonant energy transfer between the OD vibrations. We determine the Förster radius of the OD vibration of HDO in H2O to be r0 = 2.3 +/- 0.2 A.

Interfacial Structure in Thin Water Layers Formed by Forced Dewetting on Self-Assembled Monolayers of ω-Terminated Alkanethiols on Ag

A method for the spectroscopic characterization of interfacial fluid molecular structure near solid substrates is reported. The thickness and interfacial molecular structure of residual ultrathin D20 films remaining after forced dewetting on alkanethiolate self-assembled monolayers (SAMs) of 11 1-mercaptoundecanoic acid (11-MUA), 11-mercaptoundecanol (11-MUD), and undecanethiol (UDT) on Ag are investigated using ellipsometry and surface Raman spectroscopy. The residual film thickness left after withdrawal is greater on hydrophilic SAMs than on hydrophobic SAMs. This behavior is rationalized on the basis of differing degrees of fluid slip within the interfacial region due to different interfacial molecular structure. The v(O-D) regions of surface Raman spectra clearly indicate unique interfacial molecular properties within these films that differ from bulk D20. Although the residual films are created by shear forces and Marangoni flow at the three-phase line during the forced dewetting process, the nature of the films sampled optically must also be considered from the standpoint of thin film stability after dewetting. Thus, the resulting D20 films exist in vastly different morphologies depending on the nature of the water-SAM interactions. Residual D20 is proposed to exist as small nanodroplets on UDT surfaces due tospontaneous rupture of the film after dewetting. In contrast, on 11-MUD and 11-MUA surfaces, these films exist in a metastable state that retains their conformal nature on the underlying modified surface. Analysis of the peak intensity ratios of the so-called "ice-like" to "liquid-like" v(O-D) modes suggests more ice-like D20 character near 11-MUD surfaces, but more liquid-like character near 11-MUA and UDT surfaces. The creation of residual ultrathin films by forced dewetting is thus demonstrated to be a powerful method for characterizing interfacial molecular structure of fluids near a solid substrate under ambient conditions of temperature and pressure.

Heterogeneity of Water at the Phospholipid Membrane Interface

Femtosecond infrared (IR) two-color pump-probe experiments were used to investigate the nonlinear response of the D2O stretching vibration in weakly hydrated dimyristoyl-phosphatidylcholine (DMPC) membrane fragments. The vibrational lifetime is comparable to or longer than that in bulk D2O and is frequency dependent, as it decreases with increasing probe frequency. Also, the lifetime increases when the water content of the sample is lowered. The measured lifetimes range between 903 and 390 fs. A long-lived spectral feature grows in following the excitation and is attributed to photoinduced D-bond breaking. The photoproduct spectrum differs from the steady state difference Fourier transform infrared (FTIR) spectrum, showing that the full thermalization of the excitation energy happens on a much longer time scale than the time interval considered (12 ps). Further evidence of the inhomogeneous character of the water residing in the polar region of the bilayer comes from the spectral anisotropy. The water molecules absorbing on the low frequency side of the absorption band show no decay at all of the anisotropy, while an ultrafast partial decay appears when the high frequency side of the spectrum is probed. The overall behavior differs remarkably from that observed with similar experiments in bulk water and in water segregated in inverse micelles. In weakly hydrated phospholipid membranes, water molecules are present mostly as isolated species, prevalently involved in strong, rigid, and persistent hydrogen bonds with the polar groups of the bilayer molecules. This specific character appears to have a direct effect on the structural stability and thermal properties of the membrane.

Temperature and gas-phase composition measurements in supersonic flows using tunable diode laser absorption spectroscopy: The effect of condensation on the boundary-layer thickness

We used a tunable diode laser absorption spectrometer and a static-pressure probe to follow changes in temperature, vapor-phase concentration of D2O, and static pressure during condensation in a supersonic nozzle. Using the measured static-pressure ratio p/p0 and the mass fraction of the condensate g as inputs to the diabatic flow equations, we determined the area ratio (A/A*)wet and the corresponding centerline temperature of the flow during condensation. From (A/A*)wet we determined the boundary-layer displacement thickness during condensation (delta#)wet. We found that (delta#)wet first increases relative to the value of delta# in a dry expansion (delta#)Dry before becoming distinctly smaller than (delta#)Dry downstream of the condensation region. After correcting the temperature gradient across the boundary layers, the temperature determined from p/p0 and g agreed with the temperature determined by the laser-absorption measurements within our experimental error (+/-2 K), except when condensation occurred too close to the throat. The agreement between the two temperature measurements let us draw the following two conclusions. First, the differences in the temperature and mole fraction of D2O determined by the two experimental techniques, first observed in our previous study [P. Paci, Y. Zvinevich, S. Tanimura, B. E. Wyslouzil, M. Zahniser, J. Shorter, D. Nelson, and B. McManus, J. Chem. Phys. 121, 9964 (2004)], can be explained sufficiently by changes in delta# caused by the condensation of D2O, except when the phase transition occurs too close to the throat. Second, the extrapolation of the equation, which expresses the temperature dependence of the heat of vaporization of bulk D2O liquid, is a good estimate of the heat of condensation of supercooled D2O down to 210 K.

Study of the conformational change of poly(N‐isopropylacrylamide)‐grafted chains in water with neutron reflection: Molecular weight dependence at high grafting density

AbstractThe temperature‐dependent conformational change of poly(N‐isopropylacrylamide) (PNIPAM) brushes in D2O was investigated as a function of the molecular weight (M) at a high grafting density with neutron reflection. PNIPAM chains with three different M values were grafted at the same high surface density from a gold surface by atom transfer radical polymerization. A significant change in the segment concentration profile was observed for all three samples as the temperature passed through the lower critical solution temperature (∼30 °C), in contrast to previous results obtained for samples with much lower surface density. Somewhat surprisingly, the fractional change in the first moment of the segment concentration profile (〈z〉) from 20 to 41 °C was weaker with increasing M. This is contrary to the trend for systems involving only van der Waals (VDW) interactions, in which higher M chains experience larger conformational changes with change in solvent quality. Indeed, the M dependence of the first moment of the segment concentration profile for the grafted PNIPAM chains at 20 °C was much weaker than has been reported previously for dense brushes involving only VDW interactions under good solvent conditions. At 20 °C, the form of the segment concentration profile varied systematically with M. A single‐layer profile resulted for the highest M, but the profiles became more bilayer in character with decreasing M. At 41 °C, the profiles for all three samples were adequately described by a single dense layer with a smooth transition region to bulk D2O. The weak dependence of 〈z〉 on M at 20 °C and the trend from a bilayer profile at lower M to a single‐layer profile at higher M appear to be related. These results are interpreted in terms of concentration‐dependent segment–segment interactions that result in a weak attraction for high segment densities at 20 °C. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 3302–3310, 2004

The partitioning of alkanediols into SDS and DTAB micelles from NMR-PRE experiments

The p-values of a number of α,ω-alkanediols, 1,2-alkanediols, and 1,5-hexanediol and 2,5-hexanediol between D2O and the micellar phases of anionic sodium dodecylsulfate (SDS) and cationic dodecyltrimethylammonium bromide (DTAB) have been determined using the using a NMR paramagnetic relaxation enhancement experiment (NMR-PRE). From the p-values, mole-fraction based partition coefficients and Gibbs energies of transfer for the alkanediols from the bulk D2O phase to the micellar phase have been calculated. Differences in the partition coefficients of the α,ω-alkanediols and the 1,2-alkanediols are discussed in terms of the smaller Gibbs transfer energy per CH2 group for α,ω-alkanediols. For the 1,5- and the 2,5-hexanediols, the p-values are substantially smaller than for the other hexanediols in both SDS and DTAB micelles. All of these results are consistent with the location of the two hydroxyl groups on the carbon backbone making a substantial contribution to the thermodynamics of transfer of solubilizates.

Femtosecond mid-infrared spectroscopy of aqueous solvation shells

We use femtosecond two-color mid-infrared spectroscopy to study the dynamics of aqueous solutions of salts in HDO:D2O. We find that the lifetime of the O–H stretch vibration of HDO molecules in the solvation shell of the halogenic anions Cl−, Br−, and I− is much longer than the lifetime of the O–H stretch vibration of the HDO molecule in bulk D2O solution. This difference in lifetime allows for a clear separation of the response of the solvation shell from that of the bulk liquid. A detailed investigation of the spectral dynamics of the solvating HDO molecules reveals that the hydrogen-bond dynamics of these molecules are much slower than the hydrogen-bond dynamics of bulk liquid water.

Freezing of phosphocholine headgroup in fully hydrated sphingomyelin bilayers and its effect on the dynamics of nonfreezable water at subzero temperatures

Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy are applied to characterize the nonfreezable water molecules in fully hydrated D2O/sphingomyelin at temperatures below 0 degrees C. Upon cooling, DSC thermogram displays two thermal transitions peaked at -11 and -34 degrees C. The high-temperature exothermic transition corresponds to the freezing of the bulk D2O, and the low-temperature transition, which has not previously been reported, can be ascribed to the freezing of the phosphocholine headgroup in the lipid bilayer. The dynamics of nonfreezable water are also studied by 2H NMR T1 (spin-lattice relaxation time) and T2e (spin-spin relaxation time obtained by two pulse echo) measurements at 30.7 MHz and at temperatures down to -110 degrees C. The temperature dependence of the T1 relaxation time is characterized by a distinct minimum value of 2.1 +/- 0.1 ms at -30 degrees C. T2e is discontinuous at temperature around -70 degrees C, indicating another freezing-like event for the bound water at this temperature. Analysis of the relaxation data suggest that nonfreezable water undergoes both fast and slow motions at characteristic NMR time scales. The slow motions are affected when the lipid headgroup freezes.