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![Water dynamics across the cell envelope (CE) at 0.1 (black) and 200MPa (red), visualised using different isotopic contrast subtractions. (A) (Hc/Db-Db), with SS jump model fits to the overall cell envelope (0.2-1.8 Å −1 ) shown as dashed lines and restricted to the 0.2-1.0 Å −1 range (solid lines). (B) Data points for the corresponding isotopic contrast (Dc/Hb-Hb). Panels (C) and (D): isotopic contrasts [(Hc/Db-Im)-Db)] and [(Dc/Hb-Im)-Hb)] showing data points taking into account differential effects of pressure on Im compared with bulk media (Hb, Db).](profile/Paul-Mcmillan-4/publication/289571865/figure/fig3/AS:667623276675079@1536185201370/Water-dynamics-across-the-cell-envelope-CE-at-01-black-and-200MPa-red-visualised.jpg)
Water dynamics across the cell envelope (CE) at 0.1 (black) and 200MPa (red), visualised using different isotopic contrast subtractions. (A) (Hc/Db-Db), with SS jump model fits to the overall cell envelope (0.2-1.8 Å −1 ) shown as dashed lines and restricted to the 0.2-1.0 Å −1 range (solid lines). (B) Data points for the corresponding isotopic contrast (Dc/Hb-Hb). Panels (C) and (D): isotopic contrasts [(Hc/Db-Im)-Db)] and [(Dc/Hb-Im)-Hb)] showing data points taking into account differential effects of pressure on Im compared with bulk media (Hb, Db).
Source publication
Quasielastic neutron scattering (QENS) is an ideal technique for studying water transport and relaxation dynamics at pico- to nanosecond timescales and at length scales relevant to cellular dimensions. Studies of high pressure dynamic effects in live organisms are needed to understand Earth’s deep biosphere and biotechnology applications. Here we a...
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Citations
... Quasi-elastic neutron scattering (QENS) is a classical approach to the study of molecular dynamics diffusive processes. The method has been applied successfully to measurements of intracellular molecular dynamics on the pico-to nanosecond time scale on back-scattering spectrometers at ILL, Juelich, ISIS and ORNL [5][6][7][8][9][10][11]. The Fourier transform of a diffusive process is a Lorentzian function, from which can be derived a diffusion coefficient and relaxation time. ...
... The elastic window neutron QENS and elastic window neutron scattering experiments on live cells (partially reviewed in [21]) include studies on water mobility in Dead Sea archaea, in which a slow component was discovered, hypothetically associated with ion-binding structures [5], in E. coli, indicating about 90% of cytoplasmic water behaves similarly to bulk water, 10% being associated in macromolecular hydration shells [6], in Shewanella oneidensis at ambient and high pressure [7], in cancer [8,10,11,15] and brain cells [23], as well as on intracellular macromolecular dynamics in redblood cells and archaea (see below). ...
Incoherent neutron scattering experiments have produced important insights into intracellular molecular dynamics in vivo . Selected results highlight the role of water dynamics in cancer and brain cells, as well as cellular adaptation through the evolution of appropriate molecular dynamics, in order to respond to environmental challenges.
... In the case of NR, a wide range of research now routinely exploits the contrast enabled through the use of selective deuteration (Grage et al., 2011;Waldie et al., 2018Waldie et al., , 2019Waldie et al., , 2020. Deuterium labelling and reverse labelling have also been used for neutron scattering studies of the dynamics of biological macromolecules (Foglia et al., 2016), in particular when coupled with hydration water dynamics (Wood et al., 2013). In the case of solution-state NMR, deuterium labelling is essential for multidimensional heteronuclear NMR studies of proteins, especially high-molecular-weight proteins and macromolecular complexes. ...
This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris , as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.
... In both media, metabolic process were active and living bacteria residing in ice crystals continued to be viable upon subsequent release to ambient pressures (0.1 MPa) [Sharma et al. (2002)]. Foglia et al. (2016) observed a significant reduction of intracellular water dynamics in bacterial cells of Shewanella oneidensis under high pressure (200 Mpa). These results imply that pressures encountered at the depths of thick ice caps and deep crustal subsurface may not be a limiting factor for the existence of life, suggesting that deep (water and ice) layers of Enceladus might provide viable settings for life unhindered by the high pressures. ...
In this article, we present the results of an experiment carried out in a vacuum chamber during which we made multispectral images of Micrococcus sp. cells escaping an analog of the Enceladus’, the Saturnian satellite, ocean. We have added bacterial cells to the sample containing NaCl and NaHCO3, then we placed the sample in a vacuum chamber. We have imaged the evaporation process, bubble scrubbing, and then the process of bacteria emerge into the vacuum. We used a multispectral imaging technique in five optical channels: four visible and one NIR channel. By the analysis of the principal components of multispectral data and the ISO CLASS classification, we distinguished the phases of the experiment corresponding to the light scattering models (Mie solutions of Maxwell’s equations) on water vapor droplets and on the Micrococcus cells. We distinguished the blue and NIR channels as suitable for the detection of Micrococcus sp. in the water vapor.
... 17 Somewhat more recent studies have since concluded that there is only a small difference between cytosolic and bulk water. 18,19 To complicate the situation further, a recent QENS study found that cytosolic water exhibits decreased diffusion compared to bulk water, with the surprising result that this difference from bulk water behavior became even more drastic with the addition of cisplatin. 20 Despite the long history of studying the dynamics of cytosolic water, much remains unclear and unresolved. ...
Water provides a dynamic matrix in which all biochemical processes occur in living organisms. The structure and dynamics of intracellular water constitute the cornerstone for understanding all aspects of cellular function. Fundamentally, direct visualization of subcellular solvation heterogeneity is essential but remains challenging with commonly used nuclear magnetic resonance methods due to poor spatial resolution. To explore this question, we demonstrate a vibrational-shift imaging approach by combining the spectral-focusing hyperspectral stimulated Raman scattering technique with an environmentally sensitive nitrile probe. The sensing ability of a near-infrared nitrile-containing molecule is validated in the solution phase, microscopic droplets, and cellular environments. Finally, we quantitatively measure the subcellular solvation variance between the cytoplasm (29.5%, S.E. 1.8%) and the nucleus (57.3%, S.E. 1.0%), which is in good agreement with previous studies. This work sheds light on heterogeneous solvation in live systems using coherent Raman microscopy and opens up new avenues to explore environmental variance in complex systems with high spatiotemporal resolution.
... The potential habitability of icy moons has led to studies addressing possible microbial survival and growth [17][18][19][20][21][22], as well as estimates of biomass concentrations [23][24][25] in these environments. The results show that, under certain conditions (degree of convection, mantle rheology, etc.), there would be enough chemical energy to sustain an ecosystem of chemoautotrophic organisms. ...
With the discovery of the persistent jets of water being ejected to space from Enceladus, an understanding of the effect of the space environment on potential organisms and biosignatures in them is necessary for planning life detection missions. We experimentally determine the survivability of microbial cells in liquid medium when ejected into vacuum. Epifluorescence microscopy, using a lipid stain, and SEM imaging were used to interrogate the cellular integrity of E. coli after ejected through a pressurized nozzle into a vacuum chamber. The experimental samples showed a 94% decrease in visible intact E. coli cells but showed a fluorescence residue in the shape of the sublimated droplets that indicated the presence of lipids. The differences in the experimental conditions versus those expected on Enceladus should not change the analog value because the process a sample would undergo when ejected into space was representative. E. coli was selected for testing although other cell types could vary physiologically which would affect their response to a vacuum environment. More testing is needed to determine the dynamic range in concentration of cells expected to survive the plume environment. However, these results suggest that lipids may be directly detectable evidence of life in icy world plumes.
... Therefore, our understanding of the processes going on underneath the ice shell rely on laboratory or computer based experiments. These experiments include studies that examine material properties under subsurface conditions, e.g. the dynamics of ices, and high-pressure facilities used for both biotic (studies regarding microbial survival and biosignature production: Taubner et al. 2018Taubner et al. , 2019Foglia et al. 2016;Schuerger and Nicholson 2016;Hazael et al. 2017;Sharma et al. 2002) and abiotic settings (studies exploring ice-water and water-silicate interactions: Vance and Brown 2008, 2013Mantegazzi et al. 2012Mantegazzi et al. , 2013. Further, investigations into environmental gradients are performed to broaden our understanding of the physical conditions on these icy worlds (More-Mutch et al. submitted). ...
... At higher pressures above 1000 bar, microbial survival is investigated using a diamond anvil (e.g., Foglia et al. 2016;Schuerger and Nicholson 2016;Hazael et al. 2017). For example, the effect of high pressure (0.0006-0.01 Mbar) on the physiology and metabolic activity of Shewanella oneidensis and Escherichia coli was investigated (Sharma et al. 2002). ...
The icy satellites of Jupiter and Saturn are perhaps the most promising places in the Solar System regarding habitability. However, the potential habitable environments are hidden underneath km-thick ice shells. The discovery of Enceladus’ plume by the Cassini mission has provided vital clues in our understanding of the processes occurring within the interior of exooceans. To interpret these data and to help configure instruments for future missions, controlled laboratory experiments and simulations are needed. This review aims to bring together studies and experimental designs from various scientific fields currently investigating the icy moons, including planetary sciences, chemistry, (micro-)biology, geology, glaciology, etc. This chapter provides an overview of successful in situ, in silico, and in vitro experiments, which explore different regions of interest on icy moons, i.e. a potential plume, surface, icy shell, water and brines, hydrothermal vents, and the rocky core.
... 25,27 To extend and confirm this data in a meaningful context, a whole cell approach has been designed to provide a third alternative for studying the effect of high concentrations in a realistic biological context. 10,[28][29][30][31][32][33] These studies use neutron scattering to determine the dynamics inside the cells, which is the method of choice because it does not induce radiation damage to cells while it allows the separation of the molecular dynamics of cell constituents or water directly inside in vivo cells. 34 In contrast to studies on the impact of crowding on pure proteins, cell studies showed that the release of crowding constraints on proteins leads to an increase in rigidity and a decrease in HHP sensitivity. ...
The hyperthermophilic piezophile Thermococcus barophilus displays a strong stress response characterized by the accumulation of the organic osmolyte mannosylglycerate during growth under sub-optimal pressure conditions (0.1 MPa). Taking advantage of this known effect, the impact of osmolytes in piezophiles in an otherwise identical cellular context was investigated, by comparing T. barophilus cells grown under low or optimal pressures (40 MPa). Using neutron scattering techniques, we studied the molecular dynamics of live cells of T. barophilus at different pressures and temperatures. We show that in presence of osmolytes, cells present a higher diffusion coefficient of hydration water and an increase of bulk water motions at high temperature. In absence of osmolytes, the T. barophilus cellular dynamics is more responsive to high temperature and high hydrostatic pressure. These results give therefore clear evidences for a protecting effect of osmolytes on proteins.
... Therefore, neutron scattering experiments can be conducted on buried samples in relatively complex sample conditions. Experimental environments in which scattering studies have taken place include extreme pressure [1], industrial processing of foodstuffs [2], and in the case of model biological membranes, conditions like those found in vivo [3,4]. ...
Neutron scattering has significant benefits for examining the structure of protein–lipid complexes. Cold (slow) neutrons are nondamaging and predominantly interact with the atomic nucleus, meaning that neutron beams can penetrate deeply into samples, which allows for flexibility in the design of samples studied. Most importantly, there is a strong difference in neutron scattering length (i.e., scattering power) between protium (\( {}_1{}^1H \), 99.98% natural abundance) and deuterium (\( {}_1{}^2H \) or D, 0.015%). Through the mixing of H2O and D2O in the samples and in some cases the deuterium labeling of the biomolecules, components within a complex can be hidden or enhanced in the scattering signal. This enables both the overall structure and the relative distribution of components within a complex to be resolved. Lipid–protein complexes are most commonly studied using neutron reflectometry (NR) and small angle neutron scattering (SANS). In this review the methodologies to produce and examine a variety of model biological membrane systems using SANS and NR are detailed. These systems include supported lipid bilayers derived from vesicle dispersions or Langmuir–Blodgett deposition, tethered bilayer systems, membrane protein–lipid complexes and polymer wrapped lipid nanodiscs. The three key stages of any SANS/NR study on model membrane systems—sample preparation, data collection, and analysis—are described together with some background on the techniques themselves.
... Drug concentrations and incubation times were chosen according to previous results, corresponding to an optimal cytotoxic effect towards this particular neoplastic cell line 7 : 4 and 8 μM (up to ca. 2xIC 50 level), for a 48 h exposure time. A multidisciplinary experimental approach was followed, through the application of: (i) Quasi-elastic neutron scattering (QENS), suitable for directly accessing different spatially resolved dynamical processes (at subnanometer lengthscale and subnanosecond timescale), under distinct conditions, mainly for hydrogen-rich systems; (Foglia, Hazael et al., 2016, Frolich, Gabel et al., 2009, Fujiwara, Araki et al., 2016 (ii) synchrotron-radiation Fourier-transform Infrared Spectroscopy-Attenuated Total Reflectance (SR-FTIR-ATR), recognized as a cutting-edge non-destructive tool for obtaining spectral signatures of molecular components in biological samples, so as to relate structural to functional data; (Baker, Trevisan et al., 2014, Holman, Bechtel et al., 2010 (iii) synchrotron-based Extended X-ray Absorption Fine Structure (EXAFS) and X-ray Absorption Near-Edge Structure (XANES), that are methods of choice for obtaining detailed information on the local structure of bioinorganic non-crystalline materials. (Obata, Harada et al., 2009, Provost, Bouvet-Muller et al., 2009 Water supports vital biochemical processes in living organisms, and is responsible for the maintenance of the functional three-dimensional architecture of biopolymers through a tight interplay within their hydration shells. ...
A complementary structural and dynamical information on drug-DNA interplay has been achieved at a molecular level, for Pt/Pd-drugs, allowing a better understanding of their pharmacodynamic profile. The interaction of two cisplatin-like dinuclear Pt(II) and Pd(II) complexes with DNA was studied through a multidisciplinary experimental approach, using quasi-elastic neutron scattering (QENS) techniques coupled to synchrotron-based extended X-ray absorption fine structure (SR-EXAFS) and Fourier-Transform Infrared Spectroscopy-Attenuated Total Reflectance (SR-FTIR-ATR). The drug impact on DNA's dynamical profile, via its hydration layer, was provided by QENS, a drug-triggered enhanced mobility having been revealed. Additionally, an onset of anharmonicity was detected for dehydrated DNA, at room temperature. Far- and mid-infrared measurements allowed the first simultaneous detection of the drugs and its primary pharmacological target, as well as the drug-prompted changes in DNA's conformation that mediate cytotoxicity in DNA extracted from drug-exposed human triple negative breast cancer cells (MDA-MB-231), a low prognosis type of cancer. The local environment of the absorbing Pd(II) and Pt(II) centers in the drugs' adducts with adenine, guanine and glutathione was attained by EXAFS.
... The applied pressure modifies the packing and order of the solvent at the protein surface, increases the protein hydration, and promotes the penetration of water in hydrophobic cavities (16,17). This induces swelling and eventually unfolding at pressures sufficient for denaturation (18). Detailed changes in protein hydration at elevated pressures have been observed in molecular dynamics simulations of isolated proteins in solution, i.e., distortion of tetrahedral structure and stiffening of vibrational modes (19). ...
Significance
This is a combined study, which employs neutron scattering experiments and molecular dynamics simulations to compare high pressure-induced effects on collective properties of protein solutions and bulk water, to observe changes in the HB network structure of protein hydration water upon compression. Our experimental results reflect distinct properties of water at the protein–water interface, while at the same time atomistic molecular dynamics simulations allow us to analyze its pressure-dependent structural changes. This study therefore provides insights that allow establishing measurements of pressure-induced variations in collective dynamics as a probe to characterize biomolecular systems as complex as entire cells.
