Deuterium Bonds Research Articles

The structure of plastocyanin tunes the midpoint potential by restricting axial ligation of the reduced copper ion

Blue copper proteins are models for illustrating how proteins tune metal properties. Nevertheless, the mechanisms by which the protein controls the metal site remain to be fully elucidated. A hindrance is that the closed shell Cu(I) site is inaccessible to most spectroscopic analyses. Carbon deuterium (C-D) bonds used as vibrational probes afford nonperturbative, selective characterization of the key cysteine and methionine copper ligands in both redox states. The structural integrity of Nostoc plastocyanin was perturbed by disrupting potential hydrogen bonds between loops of the cupredoxin fold via mutagenesis (S9A, N33A, N34A), variably raising the midpoint potential. The C-D vibrations show little change to suggest substantial alteration to the Cu(II) coordination in the oxidized state or in the Cu(I) interaction with the cysteine ligand. They rather indicate, along with visible and NMR spectroscopy, that the methionine ligand distinctly interacts more strongly with the Cu(I) ion, in line with the increases in midpoint potential. Here we show that the protein structure determines the redox properties by restricting the interaction between the methionine ligand and Cu(I) in the reduced state.

Isotope effects on the stability of the hydrogen-bonded C2H2 NCH, H2O XH3 (X = N, P, As) and F2CH2 NH3 complexes

A computational study of three sets of binary hydrogen-bonded complexes was undertaken at the M062x/6–311++G(2d,2p) level of theory to investigate the relative stabilities of mono-deuterated isotopomers of these complexes. This work allows for a comparison of the relative magnitudes of hydrogen and deuterium bonds at low temperatures in (i) the linear and T-shaped optimized structures of C2H2 NCH, (ii) in the H2O XH3 (X = N, P, As) series, where the strength of red-shifted hydrogen bonding is systematically varied via the XH3 basicity and (iii) in the blue-shifted H-bonded F2CH2 NH3 interaction. The deuterium-bonded isotopomers predominate and this energetic preference is rationalized by reference to a simple theoretical model.

Isotope effects observed in diluted D2O/H2O mixtures identify HOD-induced low-density structures in D2O but not H2O

Normal and heavy water are solvents most commonly used to study the isotope effect. The isotope effect of a solvent significantly influences the behavior of a single molecule in a solution, especially when there are interactions between the solvent and the solute. The influence of the isotope effect becomes more significant in D2O/H2O since the hydrogen bond in H2O is slightly weaker than its counterpart (deuterium bond) in D2O. Herein, we characterize the isotope effect in a mixture of normal and heavy water on the solvation of a HOD molecule. We show that the HOD molecule affects the proximal solvent molecules, and these disturbances are much more significant in heavy water than in normal water. Moreover, in D2O, we observe the formation of low-density structures indicative of an ordering of the solvent around the HOD molecule. The qualitative differences between HOD interaction with D2O and H2O were consistently confirmed with Raman spectroscopy and NMR diffusometry.

Interaction between Heavy Water and Single-Strand DNA: A SERS Study.

The structure and function of biological macromolecules change due to intermolecular deuterium bond formation or deuterium substitution with environmental D2O. In this study, surface-enhanced Raman spectroscopy (SERS) was used to detect interaction sites between D2O and ssDNA and their action mechanisms. SERS peaks of ssDNA changed with increasing D2O proportions, and the site of action mainly involved A and G bases, whose number strengthened the interaction between sequences and D2O and hence the SERS peak intensities. Fixing the number of A and G bases prevented changes in their positions from significantly altering the map. We also identified the interaction between ssDNA sequences that easily formed a G-quadruplex structure and D2O. The amplitude of the SERS peak intensity change reflected the ssDNA structural stability and number of active sites. These findings are highly significant for exploring genetic exchanges and mutations and could be used to determine the stability and structural changes of biological macromolecules.

The impact of H/D exchange on the thermal and structural properties as well as high-pressure relaxation dynamics of melatonin

In this paper, thermal properties, atomic-scale structure, and molecular dynamics (at ambient and high pressure) of native melatonin (MLT) and its partially-deuterated derivative (MLT-d2) have been investigated. Based on infrared spectroscopy, it was shown that treating MLT with D2O causes the replacement of hydrogen atoms attached to the nitrogen by deuterium. The degree of such substitution was very high (> 99%) and the deuterated sample remained stable after exposure to the air as well as during the melting and vitrification processes. Further calorimetric studies revealed the appearance of a peculiar thermal event before the melting of crystalline MLT-d2, which was assigned by the X-ray diffraction to a local negative thermal expansion of the unit cell. Finally, the high-pressure dielectric experiments indicated a few interesting findings, including the variation in the shape of the structural relaxation peak during compression, the difference in the pressure evolution of the glass transition temperature, and the temperature dependence of activation volume for both MLT species. The variations in these parameters manifest a different impact of the compression/densification on the dynamics of hydrogen and deuterium bonds in the native and partially-deuterated MLT, respectively.

Using Molecular Dynamics to Gain Atomic-Level Insight into Bilayer Properties Measured with NMR and Neutron Spin-Echo Spectroscopy

Lipid bilayers form the core of functional membrane platforms such as cellular plasma membranes. Their dynamics are inherently related to a wide range of physical and biological processes and thus have become the subject of both experimental measurements and theoretical investigations. Nuclear magnetic resonance (NMR) in particular, is able to probe a hierarchy of lipid motions by measuring the power spectrum of thermally excited molecular fluctuations. From this spectrum of molecular noise emerge both average conformational parameters such as chain order, SCD, and dynamical descriptors such as the relaxation rate, R1, of carbon-hydrogen (or deuterium) bond fluctuations. Interestingly, these experimental observables exhibit specific functional relationships including an SCD-R1 square-law dependence, and an R1 inverse square-root frequency law. Different theories explain these functional forms, but the convoluted nature of the NMR data complicates further decoupling of the signal and testing of underlying models. Here we use atomistic molecular dynamics (MD) simulations to investigate the underpinnings of the experimental results. We calculated SCD and R1 for a series of experimentally studied bilayers composed of unsaturated or saturated lipids with different cholesterol content and at different hydration levels. The simulation results show the same functional dependencies as those observed with NMR and reveal unprecedented details of the relevant molecular mechanisms. Following a recently developed paradigm, we use the slope of the SCD-R1 square-law dependence from the simulations to calculate the bilayer bending rigidity modulus kC and explore its underlying parameter space. Comparison to kC values obtained from MD splay fluctuations and neutron spin-echo measurements allows for a closer look at the complementary applications of the techniques. Our results provide novel and clarifying insights into the nature of experimentally accessible molecular motions and their biophysical implications.

Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman Scattering of Selective Deuterium Labeling.

Polyglutamine (polyQ) diseases are a group of neurodegenerative disorders, involving the deposition of aggregation-prone proteins with long polyQ expansions. However, the cytotoxic roles of these aggregates remain highly controversial, largely due to a lack of proper tools for quantitative and nonperturbative interrogations. Common methods including in vitro biochemical, spectroscopic assays, and live-cell fluorescence imaging all suffer from certain limitations. Here, we propose coupling stimulated Raman scattering microscopy with deuterium-labeled glutamine for live-cell imaging, quantification, and spectral analysis of native polyQ aggregates with subcellular resolution. First, through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) exon1 proteins for Huntington’s disease, we achieved sensitive and specific stimulated Raman scattering (SRS) imaging of carbon–deuterium bonds (C–D) from aggregates without GFP labeling, which is commonly employed in fluorescence microscopy. We revealed that these aggregates became 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantifications, which indicate a surprising dependence of protein compositions on aggregation sizes. Our further calculations, for the first time, reported the absolute concentrations for sequestered mHtt and non-mHtt proteins within the same aggregates. Third, we adopted hyperspectral SRS for Raman spectroscopic studies of aggregate structures. By inducing a cellular heat shock response, a potential therapeutic approach for inhibiting aggregate formation, we found a possible aggregate intermediate state with changed solvation microenvironments. Our method may hence readily unveil new features and mechanistic insight of polyQ aggregates and pave the way for comprehensive in vivo investigations.

Differential adsorption of analyte and deuterated analogue onto the rotor seal of the injector in high-performance liquid chromatography/electrospray ionisation mass spectrometry.

Samples were injected onto the HPLC system via either a Valco C14W.1 internal sample injector or a Rheodyne 7125 injector, both with dissimilar Vespel rotor seals, and linked to a quadrupole time-of-flight (QTOF) mass spectrometer. Alternatively, samples were infused directly into the mass spectrometer via a syringe-driver. Electrospray ionisation in positive mode was used in both cases. Experiments demonstrated significant differential adsorption of the analyte (RAC inhibitor, NSC23766) and its tri-deuterated internal standard onto the Vespel rotor seal, with the latter seeming to be preferentially adsorbed. The deuterated analogue also showed lower electrospray sensitivity, as demonstrated by syringe infusion of a mixture of the compounds, compared with their separate infusion at the same concentration. This study has demonstrated the problem of differential adsorption onto HPLC Vespel rotor seals, and that the use of a stable-isotope-labelled analogue as internal standard does not guarantee the constancy of the analyte/internal response ratio in quantitative methods. The partial solution was to work at much higher concentration where adsorption, whilst still apparent, was relatively insignificant.

Attenuated Deuterium Stabilization of Hydrogen-Bound Complexes at Room Temperature.

We have observed nine bimolecular hydrogen- or deuterium-bound complexes at room temperature using Fourier transform infrared (FTIR) spectroscopy. The complexes were formed using methanol or ethanol as hydrogen bond donors, as well as deuterated isotopologues of these, in order to study isotopic effects on hydrogen bonds. The complexes were formed using either a dimethylether- (O) or trimethylamine (N) acceptor, to facilitate comparison of two different types of hydrogen or deuterium bonds, OH(D)·O and OH(D)·N. For each complex, the characteristic OH- or OD-stretching fundamental band in the bimolecular complex was observed. The Gibbs energy of complex formation was determined at room temperature for each complex to compare the relative stability of hydrogen- and deuterium-bound bimolecular complexes. It is well known that deuterium-bound complexes are more stable at low temperatures because of the lower frequency of its intermolecular modes and thus a lower zero point vibrational energy. However, at room temperature, entropic contributions to the stability should also be considered. At room temperature, we find the Gibbs energy of complex formation for each pair of corresponding hydrogen- and deuterium-bound complex to be similar. The similar values of the Gibbs energies at room temperature is explained from a difference in the entropy, upon complexation, which favors the formation of the hydrogen-bound complex more than the deuterium-bound complex at higher temperatures.

Involvement of Local, Rapid Conformational Dynamics in Binding of Flexible Recognition Motifs.

Flexible protein sequences populate ensembles of rapidly interconverting states differentiated by small-scale fluctuations; however, elucidating whether and how the ensembles determine function experimentally is challenged by the combined high spatial and temporal resolution needed to capture the states. We used carbon-deuterium (C-D) bond vibrations incorporated as infrared probes to characterize with residue-specific detail the heterogeneity of states adopted by proline-rich (PR) sequences and assess their involvement in recognition of Src homology 3 domains. The C-D absorption envelopes provided evidence for two or three sub-populations at all proline residues. The changes in the subpopulations induced by binding generally reflected recognition by conformational selection but depended on the residue and the state of the ligand to illuminate distinct mechanisms among the PR ligands. Notably, the spectral data indicate that greater adaptability among the states is associated with reduced recognition specificity and that perturbation to the ensemble populations contributes to differences in binding entropy. Broadly, the study quantifies rapidly interconverting ensembles with residue-specific detail and implicates them in function.

Residue-Specific Conformational Heterogeneity of Proline-Rich Sequences Uncovered via Infrared Spectroscopy.

Conformational heterogeneity is critical to understanding protein function but challenging to quantify. Experimental approaches that can provide sufficient temporal and spatial resolution to measure even rapidly interconverting states at specific locations in proteins are needed to fully elucidate the contribution of conformational heterogeneity and dynamics to function. Infrared spectroscopy in combination with the introduction of carbon deuterium bonds, which provide frequency-resolved probes of their environments, can uncover rapidly interconverting states with residue-specific detail. Using this approach, we quantify conformational heterogeneity of proline-rich peptides associated with different proline backbone conformations, as well as reveal their dependence on the sequence context.

Optical imaging of metabolic dynamics in animals

Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D2O-derived deuterium into macromolecules generates carbon–deuterium (C–D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C–D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C–D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.

Effects of source gas molecules on N–H- and N–D-related defect formations in GaAsN grown by chemical beam epitaxy

The formation mechanism of N–H-related defects in GaAsN grown by chemical beam epitaxy (CBE) is studied on the basis of the isotope effects on the local vibration modes (LVMs) originating from N–H. When deuterated monomethylhydrazine (MMHy) is used as the N source, LVM signals from the nitrogen–deuterium bond (N–D) are obtained. However, there are still N–H peaks in the IR absorption spectra, which have intensities similar to those of N–D peaks. When the film is grown with deuterated triethylgallium (TEGa), there are no N–D peaks. The peak intensity at 2952 cm−1 increases with increasing tris(dimethylamino)arsenic (TDMAAs) flow rate, and that at 3098 cm−1 is almost constant regardless of the flow rate. These results indicate that H atoms in the N–H-related defects originate from H directly bonded to N in MMHy and CH3 in MMHy and/or H in TDMAAs, not from TEGa.

Dynamics of the chemical bond: inter- and intra-molecular hydrogen bond.

In this discussion, we show that a static definition of a 'bond' is not viable by looking at a few examples for both inter- and intra-molecular hydrogen bonding. This follows from our earlier work (Goswami and Arunan, Phys. Chem. Chem. Phys. 2009, 11, 8974) which showed a practical way to differentiate 'hydrogen bonding' from 'van der Waals interaction'. We report results from ab initio and atoms in molecules theoretical calculations for a series of Rg∙∙∙HX complexes (Rg=He/Ne/Ar and X=F/Cl/Br) and ethane-1,2-diol. Results for the Rg∙∙∙HX/DX complexes show that Rg∙∙∙DX could have a 'deuterium bond' even when Rg∙∙∙HX is not 'hydrogen bonded', according to the practical criterion given by Goswami and Arunan. Results for ethane-1,2-diol show that an 'intra-molecular hydrogen bond' can appear during a normal mode vibration which is dominated by the OO stretching, though a 'bond' is not found in the equilibrium structure. This dynamical 'bond' formation may nevertheless be important in ensuring the continuity of electron density across a molecule. In the former case, a vibration 'breaks' an existing bond and in the later case, a vibration leads to 'bond' formation. In both cases, the molecule/complex stays bound irrespective of what happens to this 'hydrogen bond'. Both these cases push the borders on the recent IUPAC recommendation on hydrogen bonding (Arunan et al. Pure. Appl. Chem. 2011, 83 1637) and justify the inclusive nature of the definition.

Complex transition metal hydrides: linear correlation of countercation electronegativity versus T-D bond lengths.

For homoleptic 18-electron complex hydrides, an inverse linear correlation has been established between the T-deuterium bond length (T = Fe, Co, Ni) and the average electronegativity of the metal countercations. This relationship can be further employed towards aiding structural solutions and predicting physical properties of novel complex transition metal hydrides.

SERS imaging of cell-surface biomolecules metabolically labeled with bioorthogonal Raman reporters.

Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging.

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial-temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon-deuterium bonds (C-D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.

The Ionic Hydrogen/Deuterium Bonds between Diammoniumalkane Dications and Halide Anions.

Halide-anion binding to 1,12-dodecanediammonium, tetramethyl-1,12-dodecanediammmonium, and tetramethyl-1,7-heptanediammonium has been investigated with infrared multiple-photon dissociation (IRMPD) spectroscopy in the 1000-2250 cm-1 spectral region and with theory. Both charged ammonium groups in these diammonium compounds interact with the halide anion resulting in an ionic hydrogen bond (IHB) stretching frequency outside of the spectral frequency range that can be measured with the free-electron laser (FEL). This frequency is shifted into the spectral range upon exchanging all of the labile hydrogen atoms with deuterium atoms, thus making measurement of the ionic deuterium bond (IDB) stretching frequency possible. The IDB stretching frequency shifts to higher values with increasing halide-anion size, methylation of the ammonium groups, and alkane chain length, consistent with the halide-anion-deuterium bond strength decreasing with decreasing gas-phase basicity of the halide anion and the increasing gas-phase basicity of the ammonium groups. The IDB stretching frequency also depends on the alkane chain length owing to constraints on the angle of the bonds between the halide anion and the two ammonium groups. There are additional bands in the IDB stretching feature in the IRMPD spectra, which are attributed to Fermi resonances and arise from coupling with overtone or combination bands that can be identified from theory and depend on the halide-anion identity and alkane chain length.

Ordering of the O(2)…D… O(2) bonds near the phase transition in KD3(SeO3)2 single crystals by D nuclear magnetic resonance

AbstractDeuterium resonance investigations of KD3(SeO3)2 single crystals have been performed near the phase transition temperature T C. There are two types of deuterium bonds in these crystals with different behaviors at this phase transition. Our experimental results show that there are significant changes in the D spinlattice relaxation time T 1 at T C; the abrupt decrease in T 1 near T C can be explained by the critical slowing down of an overdamped soft pseudospin-type deuteron mode. Further, the ordering of the O(2)…D… O(2) bonds is affected by the phase transition, whereas the ordering of the O(1)-D… O(3) bonds is unaffected. The D NMR measurements also show that the D(2) deuteron disordering above T C is dynamic and not static.

Neutron scattering studies of K3H(SO4)2 and K3D(SO4)2: The particle-in-a-box model for the quantum phase transition

In the crystal of K(3)H(SO(4))(2) or K(3)D(SO(4))(2), dimers SO(4)···H···SO(4) or SO(4)···D···SO(4) are linked by strong centrosymmetric hydrogen or deuterium bonds whose O···O length is ≈2.50 Å. We address two open questions. (i) Are H or D sites split or not? (ii) Is there any structural counterpart to the phase transition observed for K(3)D(SO(4))(2) at T(c) ≈ 85.5 K, which does not exist for K(3)H(SO(4))(2)? Neutron diffraction by single-crystals at cryogenic or room temperature reveals no structural transition and no resolvable splitting of H or D sites. However, the width of the probability densities suggest unresolved splitting of the wavefunctions suggesting rigid entities H(L1/2)-H(R1/2) or D(L1/2)-D(R1/2) whose separation lengths are l(H) ≈ 0.16 Å or l(D) ≈ 0.25 Å. The vibrational eigenstates for the center of mass of H(L1/2)-H(R1/2) revealed by inelastic neutron scattering are amenable to a square-well and we suppose the same potential holds for D(L1/2)-D(R1/2). In order to explain dielectric and calorimetric measurements of mixed crystals K(3)D((1-ρ))H(ρ)(SO(4))(2) (0 ≤ ρ ≤ 1), we replace the classical notion of order-disorder by the quantum notion of discernible (e.g., D(L1/2)-D(R1/2)) or indiscernible (e.g., H(L1/2)-H(R1/2)) components depending on the separation length of the split wavefunction. The discernible-indiscernible isostructural transition at finite temperatures is induced by a thermal pure quantum state or at 0 K by ρ.