Spectroscopic evidence for a gold-coloured metallic water solution.

Insulating materials can in principle be made metallic by applying pressure. In the case of pure water, this is estimated1 to require a pressure of 48 megabar, which is beyond current experimental capabilities and may only exist in the interior of large planets or stars2-4. Indeed, recent estimates and experiments indicate that water at pressures accessible in the laboratory will at best be superionic with high protonic conductivity5, but not metallic with conductive electrons1. Here we show that a metallic water solution can be prepared by massive doping with electrons upon reacting water with alkali metals. Although analogous metallic solutions of liquid ammonia with high concentrations of solvated electrons have long been known and characterized6-9, the explosive interaction between alkali metals and water10,11 has so far only permitted the preparation of aqueous solutions with low, submetallic electron concentrations12-14. We found that the explosive behaviour of the water-alkali metal reaction can be suppressed by adsorbing water vapour at a low pressure of about 10-4 millibar onto liquid sodium-potassium alloy drops ejected into a vacuum chamber. This set-up leads to the formation of a transient gold-coloured layer of a metallic water solution covering the metal alloy drops. The metallic character of this layer, doped with around 5 × 1021 electrons per cubic centimetre, is confirmed using optical reflection and synchrotron X-ray photoelectron spectroscopies.

The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization.

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

Stability and Reactivity of Aromatic Radical Anions in Solution with Relevance to Birch Reduction.

We investigate the electronic structure of aromatic radical anions in the solution phase employing a combination of liquid-jet (LJ) photoelectron (PE) spectroscopy measurements and electronic structure calculations. By using recently developed protocols, we accurately determine the vertical ionization energies of valence electrons of both the solvent and the solute molecules. In particular, we first characterize the pure solvent of tetrahydrofuran (THF) by LJ-PE measurements in conjunction with ab initio molecular dynamics simulations and G0W0 calculations. Next, we determine the electronic structure of neutral naphthalene (Np) and benzophenone (Bp) as well as their radical anion counterparts Np- and Bp- in THF. Wherever feasible, we performed orbital assignments of the measured PE features of the aromatic radical anions, with comparisons to UV-vis absorption spectra of the corresponding neutral molecules being instrumental in rationalizing the assignments. Analysis of the electronic structure differences between the neutral species and their anionic counterparts provides understanding of the primarily electrostatic stabilization of the radical anions in solution. Finally, we obtain a very good agreement of the reduction potentials extracted from the present LJ-PES measurements of Np- and Bp- in THF with previous electrochemical data from cyclic voltammetry measurements. In this context, we discuss how the choice of solvent holds significant implications for optimizing conditions for the Birch reduction process, wherein aromatic radical anions play crucial roles as reactive intermediates.

Hydration of biologically relevant tetramethylammonium cation by neutron scattering and molecular dynamics.

Neutron scattering and molecular dynamics studies were performed on a concentrated aqueous tetramethylammonium (TMA) chloride solution to gain insight into the hydration shell structure of TMA, which is relevant for understanding its behavior in biological contexts of, e.g., properties of phospholipid membrane headgroups or interactions between DNA and histones. Specifically, neutron diffraction with isotopic substitution experiments were performed on TMA and water hydrogens to extract the specific correlation between hydrogens in TMA (HTMA) and hydrogens in water (HW). Classical molecular dynamics simulations were performed to help interpret the experimental neutron scattering data. Comparison of the hydration structure and simulated neutron signals obtained with various force field flavors (e.g. overall charge, charge distribution, polarity of the CH bonds and geometry) allowed us to gain insight into how sensitive the TMA hydration structure is to such changes and how much the neutron signal can capture them. We show that certain aspects of the hydration, such as the correlation of the hydrogen on TMA to hydrogen on water, showed little dependence on the force field. In contrast, other correlations, such as the ion-ion interactions, showed more marked changes. Strikingly, the neutron scattering signal cannot discriminate between different hydration patterns. Finally, ab initio molecular dynamics was used to examine the three-dimensional hydration structure and thus to benchmark force field simulations. Overall, while neutron scattering has been previously successfully used to improve force fields, in the particular case of TMA we show that it has only limited value to fully determine the hydration structure, with other techniques such as ab initio MD being of a significant help.

Hydration numbers of biologically relevant divalent metal cations from ab initio molecular dynamics and continuum solvation methods.

Hydration and, in particular, the coordination number of a metal ion is of paramount importance as it defines many of its (bio)physicochemical properties. It is not only essential for understanding its behavior in aqueous solutions but also determines the metal ion reference state and its binding energy to (bio)molecules. In this paper, for divalent metal cations Ca2+, Cd2+, Cu2+, Fe2+, Hg2+, Mg2+, Ni2+, Pb2+, and Zn2+, we compare two approaches for predicting hydration numbers: (1) a mixed explicit/continuum DFT-D3//COSMO-RS solvation model and (2) density functional theory based ab initio molecular dynamics. The former approach is employed to calculate the Gibbs free energy change for the sequential hydration reactions, starting from [M(H2O)2]2+ aqua complexes to [M(H2O)9]2+, allowing explicit water molecules to bind in the first or second coordination sphere and determining the most stable [M(H2O)n]2+ structure. In the latter approach, the hydration number is obtained by integrating the ion-water radial distribution function. With a couple of exceptions, the metal ion hydration numbers predicted by the two approaches are in mutual agreement, as well as in agreement with the experimental data.

Bridging Electrochemistry and Photoelectron Spectroscopy in the Context of Birch Reduction: Detachment Energies and Redox Potentials of Electron, Dielectron, and Benzene Radical Anion in Liquid Ammonia.

Birch reduction is a time-proven way to hydrogenate aromatic hydrocarbons (such as benzene), which relies on the reducing power of electrons released from alkali metals into liquid ammonia. We have succeeded to characterize the key intermediates of the Birch reduction process─the solvated electron and dielectron and the benzene radical anion─using cyclic voltammetry and photoelectron spectroscopy, aided by electronic structure calculations. In this way, we not only quantify the electron binding energies of these species, which are decisive for the mechanism of the reaction, but also use Birch reduction as a case study to directly connect the two seemingly unrelated experimental techniques.

Ionic Strength and Solution Composition Dictate the Adsorption of Cell-Penetrating Peptides onto Phosphatidylcholine Membranes.

Adsorption of arginine-rich positively charged peptides onto neutral zwitterionic phosphocholine (PC) bilayers is a key step in the translocation of those potent cell-penetrating peptides into the cell interior. In the past, we have shown both theoretically and experimentally that polyarginines adsorb to the neutral PC-supported lipid bilayers in contrast to polylysines. However, comparing our results with previous studies showed that the results often do not match even at the qualitative level. The adsorption of arginine-rich peptides onto 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) may qualitatively depend on the actual experimental conditions where binding experiments have been performed. In this work, we systematically studied the adsorption of R9 and K9 peptides onto the POPC bilayer, aided by molecular dynamics (MD) simulations and fluorescence cross-correlation spectroscopy (FCCS) experiments. Using MD simulations, we tested a series of increasing peptide concentrations, in parallel with increasing Na+ and Ca2+ salt concentrations, showing that the apparent strength of adsorption of R9 decreases upon the increase of peptide or salt concentration in the system. The key result from the simulations is that the salt concentrations used experimentally can alter the picture of peptide adsorption qualitatively. Using FCCS experiments with fluorescently labeled R9 and K9, we first demonstrated that the binding of R9 to POPC is tighter by almost 2 orders of magnitude compared to that of K9. Finally, upon the addition of an excess of either Na+ or Ca2+ ions with R9, the total fluorescence correlation signal is lost, which implies the unbinding of R9 from the PC bilayer, in agreement with our predictions from MD simulations.

Electronic structure of the solvated benzene radical anion.

The benzene radical anion is a molecular ion pertinent to several organic reactions, including the Birch reduction of benzene in liquid ammonia. The species exhibits a dynamic Jahn-Teller effect due to its open-shell nature and undergoes pseudorotation of its geometry. Here, we characterize the complex electronic structure of this condensed-phase system based on ab initio molecular dynamics simulations and GW calculations of the benzene radical anion solvated in liquid ammonia. Using detailed analysis of the molecular and electronic structure, we find that the spatial character of the excess electron of the solvated radical anion follows the underlying Jahn-Teller distortions of the molecular geometry. We decompose the electronic density of states to isolate the contribution of the solute and to examine the response of the solvent to its presence. Our findings show the correspondence between instantaneous molecular structure and spin density; provide important insights into the electronic stability of the species, revealing that it is, indeed, a bound state in the condensed phase; and offer electronic densities of states that aid in the interpretation of experimental photoelectron spectra.

Gas phase C6H6 - anion: Electronic stabilization by opening of the benzene ring.

It is well established that an isolated benzene radical anion is not electronically stable. In the present study, we experimentally show that electron attachment to benzene clusters leads to weak albeit unequivocal occurrence of a C6H6 - moiety. We propose here-based on electronic structure calculation-that this moiety actually corresponds to linear structures formed by the opening of the benzene ring via electron attachment. The cluster environment is essential in this process since it quenches the internal energy released upon ring opening, which in the gas phase leads to further dissociation of this anion.

Benzene Radical Anion Microsolvated in Ammonia Clusters: Modeling the Transition from an Unbound Resonance to a Bound Species.

The benzene radical anion, well-known in organic chemistry as the first intermediate in the Birch reduction of benzene in liquid ammonia, exhibits intriguing properties from the point of view of quantum chemistry. Notably, it has the character of a metastable shape resonance in the gas phase, while measurements in solution find it to be experimentally detectable and stable. In this light, our previous calculations performed in bulk liquid ammonia explicitly reveal that solvation leads to stabilization. Here, we focus on the transition of the benzene radical anion from an unstable gas-phase ion to a fully solvated bound species by explicit ionization calculations of the radical anion solvated in molecular clusters of increasing size. The computational cost of the largest systems is mitigated by combining density functional theory with auxiliary methods including effective fragment potentials or approximating the bulk by polarizable continuum models. Using this methodology, we obtain the cluster size dependence of the vertical binding energy of the benzene radical anion converging to the value of -2.3 eV at a modest computational cost.

Anisotropic diffusion of membrane proteins at experimental timescales.

Single-particle tracking (SPT) experiments of lipids and membrane proteins provide a wealth of information about the properties of biomembranes. Careful analysis of SPT trajectories can reveal deviations from ideal Brownian behavior. Among others, this includes confinement effects and anomalous diffusion, which are manifestations of both the nanoscale structure of the underlying membrane and the structure of the diffuser. With the rapid increase in temporal and spatial resolution of experimental methods, a new aspect of the motion of the particle, namely, anisotropic diffusion, might become relevant. This aspect that so far received only little attention is the anisotropy of the diffusive motion and may soon provide an additional proxy to the structure and topology of biomembranes. Unfortunately, the theoretical framework for detecting and interpreting anisotropy effects is currently scattered and incomplete. Here, we provide a computational method to evaluate the degree of anisotropy directly from molecular dynamics simulations and also point out a way to compare the obtained results with those available from SPT experiments. In order to probe the effects of anisotropic diffusion, we performed coarse-grained molecular dynamics simulations of peripheral and integral membrane proteins in flat and curved bilayers. In agreement with the theoretical basis, our computational results indicate that anisotropy can persist up to the rotational relaxation time [τ=(2Dr)-1], after which isotropic diffusion is observed. Moreover, the underlying topology of the membrane bilayer can couple with the geometry of the particle, thus extending the spatiotemporal domain over which this type of motion can be detected.

Arginine-rich cell-penetrating peptides induce membrane multilamellarity and subsequently enter via formation of a fusion pore.

Arginine-rich cell-penetrating peptides do not enter cells by directly passing through a lipid membrane; they instead passively enter vesicles and live cells by inducing membrane multilamellarity and fusion. The molecular picture of this penetration mode, which differs qualitatively from the previously proposed direct mechanism, is provided by molecular dynamics simulations. The kinetics of vesicle agglomeration and fusion by an iconic cell-penetrating peptide-nonaarginine-are documented via real-time fluorescence techniques, while the induction of multilamellar phases in vesicles and live cells is demonstrated by a combination of electron and fluorescence microscopies. This concert of experiments and simulations reveals that the identified passive cell penetration mechanism bears analogy to vesicle fusion induced by calcium ions, indicating that the two processes may share a common mechanistic origin.

Tail-Oxidized Cholesterol Enhances Membrane Permeability for Small Solutes.

Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7ß-hydroxycholesterol (7ß-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane-bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7ß-OH-chol which represents ring-oxidized sterols.

Simulation of Raman and Raman optical activity of saccharides in solution.

Structural studies of sugars in solution are challenging for most of the traditional analytical techniques. Raman and Raman optical activity (ROA) spectroscopies were found to be extremely convenient for this purpose. However, Raman and ROA spectra of saccharides are challenging to interpret and model due to saccharides' flexibility and polarity. In this study, we present an optimized computational protocol that enables the simulation of the spectra efficiently. Our protocol, which results in good agreement with experiments, combines molecular dynamics and density functional theory calculations. It further uses a smart optimization procedure and a novel adaptable scaling function. The numerical stability and accuracy of individual computational steps are evaluated by comparing simulated and experimental spectra of d-glucose, d-glucuronic acid, N-acetyl-d-glucosamine, methyl ß-d-glucopyranoside, methyl ß-d-glucuronide, and methyl ß-N-acetyl-d-glucosaminide. Overall, our Raman and ROA simulation protocol allows one to routinely and reliably calculate the spectra of small saccharides and opens the door to advanced applications, such as complete 3-dimensional structural determination by direct interpretation of the experimental spectra.

Binding of divalent cations to acetate: molecular simulations guided by Raman spectroscopy.

In spite of the biological importance of the binding of Zn2+, Ca2+, and Mg2+ to the carboxylate group, cation-acetate binding affinities and binding modes remain actively debated. Here, we report the first use of Raman multivariate curve resolution (Raman-MCR) vibrational spectroscopy to obtain self-consistent free and bound metal acetate spectra and one-to-one binding constants, without the need to invoke any a priori assumptions regarding the shapes of the corresponding vibrational bands. The experimental results, combined with classical molecular dynamics simulations with a force field effectively accounting for electronic polarization via charge scaling and ab initio simulations, indicate that the measured binding constants pertain to direct (as opposed to water separated) ion pairing. The resulting binding constants do not scale with cation size, as the binding constant to Zn2+ is significantly larger than that to either Mg2+ or Ca2+, although Zn2+ and Mg2+ have similar radii that are about 25% smaller than Ca2+. Remaining uncertainties in the metal acetate binding free energies are linked to fundamental ambiguities associated with identifying the range of structures pertaining to non-covalently bound species.

Self-association of a highly charged arginine-rich cell-penetrating peptide.

Small-angle X-ray scattering (SAXS) measurements reveal a striking difference in intermolecular interactions between two short highly charged peptides-deca-arginine (R10) and deca-lysine (K10). Comparison of SAXS curves at high and low salt concentration shows that R10 self-associates, while interactions between K10 chains are purely repulsive. The self-association of R10 is stronger at lower ionic strengths, indicating that the attraction between R10 molecules has an important electrostatic component. SAXS data are complemented by NMR measurements and potentials of mean force between the peptides, calculated by means of umbrella-sampling molecular dynamics (MD) simulations. All-atom MD simulations elucidate the origin of the R10-R10 attraction by providing structural information on the dimeric state. The last two C-terminal residues of R10 constitute an adhesive patch formed by stacking of the side chains of two arginine residues and by salt bridges formed between the like-charge ion pair and the C-terminal carboxyl groups. A statistical analysis of the Protein Data Bank reveals that this mode of interaction is a common feature in proteins.

Valence and Core-Level X-ray Photoelectron Spectroscopy of a Liquid Ammonia Microjet.

Photoelectron spectroscopy of microjets expanded into vacuum allows access to orbital energies for solute or solvent molecules in the liquid phase. Microjets of water, acetonitrile and alcohols have previously been studied; however, it has been unclear whether jets of low temperature molecular solvents could be realized. Here we demonstrate a stable 20 µm jet of liquid ammonia (-60 °C) in a vacuum, which we use to record both valence and core-level band photoelectron spectra using soft X-ray synchrotron radiation. Significant shifts from isolated ammonia in the gas-phase are observed, as is the liquid-phase photoelectron angular anisotropy. Comparisons with spectra of ammonia in clusters and the solid phase, as well as spectra for water in various phases potentially reveal how hydrogen bonding is reflected in the condensed phase electronic structure.

Arginine "Magic": Guanidinium Like-Charge Ion Pairing from Aqueous Salts to Cell Penetrating Peptides.

It is a textbook knowledge that charges of the same polarity repel each other. For two monovalent ions in the gas phase at a close contact this repulsive interaction amounts to hundreds of kilojoules per mole. In aqueous solutions, however, this Coulomb repulsion is strongly attenuated by a factor equal to the dielectric constant of the medium. The residual repulsion, which now amounts only to units of kilojoules per mole, may be in principle offset by attractive interactions. Probably the smallest cationic pair, where a combination of dispersion and cavitation forces overwhelms the Coulomb repulsion, consists of two guanidinium ions in water. Indeed, by a combination of molecular dynamics with electronic structure calculations and electrophoretic, as well as spectroscopic, experiments, we have demonstrated that aqueous guanidinium cations form (weakly) thermodynamically stable like-charge ion pairs. The importance of pairing of guanidinium cations in aqueous solutions goes beyond a mere physical curiosity, since it has significant biochemical implications. Guanidinium chloride is known to be an efficient and flexible protein denaturant. This is due to the ability of the orientationally amphiphilic guanidinium cations to disrupt various secondary structural motifs of proteins by pairing promiscuously with both hydrophobic and hydrophilic groups, including guanidinium-containing side chains of arginines. The fact that the cationic guanidinium moiety forms the dominant part of the arginine side chain implies that the like-charge ion pairing may also play a role for interactions between peptides and proteins. Indeed, arginine-arginine pairing has been frequently found in structural protein databases. In particular, when strengthened by a presence of negatively charged glutamate, aspartate, or C-terminal carboxylic groups, this binding motif helps to stabilize peptide or protein dimers and is also found in or near active sites of several enzymes. The like-charge pairing of the guanidinium side-chain groups may also hold the key to the understanding of the arginine "magic", that is, the extraordinary ability of arginine-rich polypeptides to passively penetrate across cellular membranes. Unlike polylysines, which are also highly cationic but lack the ease in crossing membranes, polyarginines do not exhibit mutual repulsion. Instead, they accumulate at the membrane, weaken it, and might eventually cross in a concerted, "train-like" manner. This behavior of arginine-rich cell penetrating peptides can be exploited when devising smart strategies how to deliver in a targeted way molecular cargos into the cell.

Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules.

Human insulin is a pivotal protein hormone controlling metabolism, growth, and aging and whose malfunctioning underlies diabetes, some cancers, and neurodegeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available and fall into three conformational states: T6, T3Rf3, and R6 As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic ß-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state-specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations.

Calcium ions in aqueous solutions: Accurate force field description aided by ab initio molecular dynamics and neutron scattering.

We present a combination of force field and ab initio molecular dynamics simulations together with neutron scattering experiments with isotopic substitution that aim at characterizing ion hydration and pairing in aqueous calcium chloride and formate/acetate solutions. Benchmarking against neutron scattering data on concentrated solutions together with ion pairing free energy profiles from ab initio molecular dynamics allows us to develop an accurate calcium force field which accounts in a mean-field way for electronic polarization effects via charge rescaling. This refined calcium parameterization is directly usable for standard molecular dynamics simulations of processes involving this key biological signaling ion.