Insights into the solubility of γ D-crystallin from multiscale atomistic simulations.

The molecular basis underlying the rich phase behavior of globular proteins remains poorly understood. We use atomistic multiscale molecular simulations to model the solution-state conformational dynamics and interprotein interactions of γ D-crystallin and its P23T-R36S mutant, which drastically limits the protein solubility, at both infinite dilution and at a concentration where the mutant fluid phase and crystalline phase coexist. We find that while the mutant conserves the protein fold, changes to the surface exposure of residues in the neighborhood of residue-36 enhance protein-protein interactions and develop specific protein-protein contacts found in the protein crystal lattice.

Adsorption of 6-MHO on two indoor relevant surface materials: SiO2 and TiO2.

The compound 6-methyl-5-hepten-2-one (6-MHO) is a product of skin oil ozonolysis and is of significance in understanding the role of human occupants in the indoor environment. We present a joint computational and experimental study investigating the adsorption of 6-MHO on two model indoor relevant surfaces, SiO2, a model for a glass window, and TiO2, a component of paint and self-cleaning surfaces. Our classical force field-based molecular dynamics, ab initio molecular dynamics simulations, and FTIR absorption spectra indicate 6-MHO can adsorb on to both of these surfaces via hydrogen and π-hydrogen bonds and is quite stable due to the linear geometry of 6-MHO. Detailed analysis of 6-MHO on the SiO2 surface shows that relative humidity does not impact surface adsorption and adsorbed water does not displace 6-MHO from the hydroxylated SiO2 surface. Additionally, the desorption kinetics of 6-MHO from the hydroxylated SiO2 surface is compared to other compounds found in indoor environments and 6-MHO is shown to desorb with a first order rate constant that is approximately four times slower than that of limonene, but six times faster than that of carvone. In addition, our joint results indicate 6-MHO forms a stronger interaction with the TiO2 surface compared to the SiO2 surface. This study suggests that skin oil ozonolysis products can partition to indoor surfaces leading to the formation of organic films.

Voltage-dependent structural models of the human Hv1 proton channel from long-timescale molecular dynamics simulations.

The voltage-gated Hv1 proton channel is a ubiquitous membrane protein that has roles in a variety of cellular processes, including proton extrusion, pH regulation, production of reactive oxygen species, proliferation of cancer cells, and increased brain damage during ischemic stroke. A crystal structure of an Hv1 construct in a putative closed state has been reported, and structural models for the channel open state have been proposed, but a complete characterization of the Hv1 conformational dynamics under an applied membrane potential has been elusive. We report structural models of the Hv1 voltage-sensing domain (VSD), both in a hyperpolarized state and a depolarized state resulting from voltage-dependent conformational changes during a 10-µs-timescale atomistic molecular dynamics simulation in an explicit membrane environment. In response to a depolarizing membrane potential, the S4 helix undergoes an outward displacement, leading to changes in the VSD internal salt-bridge network, resulting in a reshaping of the permeation pathway and a significant increase in hydrogen bond connectivity throughout the channel. The total gating charge displacement associated with this transition is consistent with experimental estimates. Molecular docking calculations confirm the proposed mechanism for the inhibitory action of 2-guanidinobenzimidazole (2GBI) derived from electrophysiological measurements and mutagenesis. The depolarized structural model is also consistent with the formation of a metal bridge between residues located in the core of the VSD. Taken together, our results suggest that these structural models are representative of the closed and open states of the Hv1 channel.

Multiphase Ozonolysis of Oleic Acid-Based Lipids: Quantitation of Major Products and Kinetic Multilayer Modeling.

Commonly found in atmospheric aerosols, cooking oils, and human sebum, unsaturated lipids rapidly decay upon exposure to ozone, following the Criegee mechanism. Here, the gas-surface ozonolysis of three oleic acid-based compounds was studied in a reactor and indoors. Under dry conditions, quantitative product analyses by 1H NMR indicate up to 79% molar yield of stable secondary ozonides (SOZs) in oxidized triolein and methyl oleate coatings. Elevated relative humidity (RH) significantly suppresses the SOZ yields, enhancing the formation of condensed-phase aldehydes and volatile C9 products. Along with kinetic parameters informed by molecular dynamics simulations, these results were used as constraints in a kinetic multilayer model (KM-GAP) simulating triolein ozonolysis. Covering a wide range of coating thicknesses and ozone levels, the model predicts a much faster decay near the gas-lipid interface compared to the bulk. Although the dependence of RH on SOZ yields is well predicted, the model overestimates the production of H2O2 and aldehydes. With negligible dependence on RH, the product composition for oxidized oleic acid is substantially affected by a competitive reaction between Criegee intermediates (CIs) and carboxylic acids. The resulting α-acyloxyalkyl hydroperoxides (α-AAHPs) have much higher molar yields (29-38%) than SOZs (12-16%). Overall, the ozone-lipid chemistry could affect the indoor environment through "crust" accumulation on surfaces and volatile organic compound (VOC) emission. In the atmosphere, the peroxide formation and changes in particle hygroscopicity may have effects on climate. The related health impacts are also discussed.

Interactions of limonene and carvone on titanium dioxide surfaces.

Limonene, a monoterpene, found in cleaning products and air fresheners can interact with a variety of surfaces in indoor environments. An oxidation product of limonene, carvone, has been reported to cause contact allergens. In this study, we have investigated the interactions of limonene and carvone with TiO2, a component of paint and self-cleaning surfaces, at 297 ± 1 K with FTIR spectroscopy and force field-based molecular dynamics and ab initio simulations. The IR absorption spectra and computational methods show that limonene forms π-hydrogen bonds with the surface O-H groups on the TiO2 surface and that carvone adsorbs on the TiO2 surface through a variety of molecular interactions including through carbonyl oxygen atoms with Ti4+ surface atoms, O-H hydrogen bonding (carbonyl O⋯HO) and π-hydrogen bonds with surface O-H groups. Furthermore, we investigated the effects of relative humidity (RH) on the adsorption of limonene and carvone on the TiO2 surface. The spectroscopic results show that the adsorbed limonene can be completely displaced by water at a relative humidity of ca. 50% RH (∼2 MLs of water) and that 25% of carvone is displaced at ca. 67% RH, which agrees with the calculated free energies of adsorption which show carvone more strongly adsorbs on the surface relative to limonene and thus would be harder to displace from the surface. Overall, this study shows how a monoterpene and its oxidation product interact with TiO2 and the impact of relative humidity on these interactions.

Thermodynamics and Mechanism of the Membrane Permeation of Hv1 Channel Blockers.

The voltage-gated proton channel Hv1 mediates efflux of protons from the cell. Hv1 integrally contributes to various physiological processes including pH homeostasis and the respiratory burst of phagocytes. Inhibition of Hv1 may provide therapeutic avenues for the treatment of inflammatory diseases, breast cancer, and ischemic brain damage. In this work, we investigate two prototypical Hv1 inhibitors, 2-guanidinobenzimidazole (2GBI), and 5-chloro-2-guanidinobenzimidazole (GBIC), from an experimentally screened class of guanidine derivatives. Both compounds block proton conduction by binding the same site located on the intracellular side of the channel. However, when added to the extracellular medium, the compounds strongly differ in their ability to inhibit proton conduction, suggesting substantial differences in membrane permeability. Here, we compute the potential of mean force for each compound to permeate through the membrane using atomistic molecular dynamics simulations with the adaptive biasing force method. Our results rationalize the putative distinction between these two blockers with respect to their abilities to permeate the cellular membrane.

Diffusivelike Motions in a Solvent-Free Protein-Polymer Hybrid.

The interaction between proteins and hydration water stabilizes protein structure and promotes functional dynamics, with water translational motions enabling protein flexibility. Engineered solvent-free protein-polymer hybrids have been shown to preserve protein structure, function, and dynamics. Here, we used neutron scattering, protein and polymer perdeuteration, and molecular dynamics simulations to explore how a polymer dynamically replaces water. Even though relaxation rates and vibrational properties are strongly modified in polymer coated compared to hydrated proteins, liquidlike polymer dynamics appear to plasticize the conjugated protein in a qualitatively similar way as do hydration-water translational motions.

Experimental and Simulation Studies of Aquaporin 0 Water Permeability and Regulation.

We begin with the history of aquaporin zero (AQP0), the most prevalent membrane protein in the eye lens, from the early days when AQP0 was a protein of unknown function known as Major Intrinsic Protein 26. We progress through its joining the aquaporin family as a water channel in its own right and discuss how regulation of its water permeability by pH and calcium came to be discovered experimentally and linked to lens homeostasis and development. We review the development of molecular dynamics (MD) simulations of lipid bilayers and membrane proteins, including aquaporins, with an emphasis on simulation studies that have elucidated the mechanisms of water conduction, selectivity, and proton exclusion by aquaporins in general. We also review experimental and theoretical progress toward understanding why mammalian AQP0 has a lower water permeability than other aquaporins and the evolution of our present understanding of how its water permeability is regulated by pH and calcium. Finally, we discuss how MD simulations have elucidated the nature of lipid interactions with AQP0.

Heterogeneous Interactions of Prevalent Indoor Oxygenated Organic Compounds on Hydroxylated SiO2 Surfaces.

Oxygenated organic compounds (OOCs) are widely found in indoor environments and come from either the direct emissions from indoor activities or the subsequent oxidation of nonoxygenated OCs. Adsorption and partitioning of OCs on surfaces are significant processes in indoor chemistry, yet these interactions specifically involving OOCs are still poorly understood. In this study, we investigate the interactions of three prevalent indoor OOCs (dihydromyrcenol, α-terpineol, and linalool) on an indoor surface proxy (hydroxylated SiO2) by combining vibrational spectroscopy with ab initio molecular dynamics simulations. The adsorption of these compounds on the SiO2 surface is driven by π hydrogen bonding and O-H hydrogen bonding interactions, with O-H hydrogen bonding interactions being stronger. The results of kinetic measurements suggest that indoor surfaces play a significant role in the removal of these OOCs, especially under moderate and low air exchange. Additionally, indoor surfaces can also serve as a reservoir of OOCs due to their much slower desorption kinetics when compared to other indoor relevant organic compounds such as limonene. Overall, the results gleaned by experiment and theoretical simulations provide a molecular representation of the interaction of OOCs on indoor relevant surfaces as well as implications of these interactions for indoor air chemistry.

Adsorption of constitutional isomers of cyclic monoterpenes on hydroxylated silica surfaces.

We present a study of four monoterpene isomers (limonene, γ-terpinene, terpinolene, and α-pinene) that are prevalent in indoor environments and their interaction with the hydroxylated SiO2 surface, a model for the glass surface, by combining infrared spectroscopy and computational simulations. These isomers are molecularly adsorbed onto SiO2 through π-hydrogen bonds with surface hydroxyl groups. However, experimental results suggest that the strength of interaction of these compounds with the SiO2 surface varies for each isomer, with α-pinene showing the weakest interaction. This observation is supported by molecular dynamics simulations that α-pinene adsorbed on the SiO2 surface has lower free energy of desorption and a lower mass accommodation coefficient compared to other isomers. Additionally, our ab initio molecular dynamics simulations show lower π-hydrogen bonding probabilities for α-pinene compared to the other three constitutional isomers. Importantly, these interactions are most likely present for a range of other systems involving organic compounds and solid surfaces and, thus, provide a thorough framework for comparing the interactions of organic molecules on indoor relevant surfaces.

Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL.

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.

Impact of Adsorbed Water on the Interaction of Limonene with Hydroxylated SiO2: Implications of π-Hydrogen Bonding for Surfaces in Humid Environments.

The indoor environment is a dynamic one with many variables impacting indoor air quality and indoor air chemistry. These include relative humidity (RH) and the presence of different surfaces. Although it has been suggested that the indoor concentrations of gas-phase compounds increase at higher relative humidity, because of displacement of these compounds from indoor surfaces, little is known from a molecular perspective about how RH and adsorbed water impact the adsorption of indoor relevant organic compounds such as limonene with indoor relevant surfaces. Herein, we investigate the effects of RH on the adsorption of limonene, a hydrophobic molecule, on hydroxylated SiO2 surfaces, a model for glass surfaces. Experimental data using infrared spectroscopy to directly measure limonene adsorption are combined with both force field-based molecular dynamics (MD) and ab initio molecular dynamics (AIMD) simulations to understand the competitive interactions between limonene, water, and the SiO2 surface. The spectroscopic data provide evidence that adsorbed limonene is not completely displaced by adsorbed water, even at high RH (∼80%) when the water layer coverage is close to three monolayers (MLs). These experimental data are supported by AIMD and MD simulations, which indicate that limonene is present at the adsorbed water interface but displaced from direct interactions with SiO2. This study shows that although some limonene can desorb from the surface, even at the highest RH, more than half the limonene remains adsorbed on the surface that can undergo continued surface reactivity. A complex network of π-hydrogen bonds, water-water hydrogen bonds, and SiO2-water hydrogen bonds explains these interactions at the air/adsorbed water/SiO2 interface that hold the hydrophobic limonene molecule at the interface. Importantly, these interactions are most likely present for a range of other systems involving organic compounds and solid surfaces at ambient relative humidity and may be important in a range of scientific areas, from sensor development to cultural heritage science.

Specific cation effects at aqueous solution-vapor interfaces: Surfactant-like behavior of Li+ revealed by experiments and simulations.

It is now well established by numerous experimental and computational studies that the adsorption propensities of inorganic anions conform to the Hofmeister series. The adsorption propensities of inorganic cations, such as the alkali metal cations, have received relatively little attention. Here we use a combination of liquid-jet X-ray photoelectron experiments and molecular dynamics simulations to investigate the behavior of K+ and Li+ ions near the interfaces of their aqueous solutions with halide ions. Both the experiments and the simulations show that Li+ adsorbs to the aqueous solution-vapor interface, while K+ does not. Thus, we provide experimental validation of the "surfactant-like" behavior of Li+ predicted by previous simulation studies. Furthermore, we use our simulations to trace the difference in the adsorption of K+ and Li+ ions to a difference in the resilience of their hydration shells.

Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF ß receptor in an active dimeric conformation.

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor ß receptor (PDGFßR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFßR complex. In this model, an active dimer of the PDGFßR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFßR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFßR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.

Voltage-Dependent Profile Structures of a Kv-Channel via Time-Resolved Neutron Interferometry.

Available experimental techniques cannot determine high-resolution three-dimensional structures of membrane proteins under a transmembrane voltage. Hence, the mechanism by which voltage-gated cation channels couple conformational changes within the four voltage sensor domains, in response to either depolarizing or polarizing transmembrane voltages, to opening or closing of the pore domain's ion channel remains unresolved. Single-membrane specimens, composed of a phospholipid bilayer containing a vectorially oriented voltage-gated K+ channel protein at high in-plane density tethered to the surface of an inorganic multilayer substrate, were developed to allow the application of transmembrane voltages in an electrochemical cell. Time-resolved neutron reflectivity experiments, enhanced by interferometry enabled by the multilayer substrate, were employed to provide directly the low-resolution profile structures of the membrane containing the vectorially oriented voltage-gated K+ channel for the activated, open and deactivated, closed states of the channel under depolarizing and hyperpolarizing transmembrane voltages applied cyclically. The profile structures of these single membranes were dominated by the voltage-gated K+ channel protein because of the high in-plane density. Importantly, the use of neutrons allowed the determination of the voltage-dependent changes in both the profile structure of the membrane and the distribution of water within the profile structure. These two key experimental results were then compared to those predicted by three computational modeling approaches for the activated, open and deactivated, closed states of three different voltage-gated K+ channels in hydrated phospholipid bilayer membrane environments. Of the three modeling approaches investigated, only one state-of-the-art molecular dynamics simulation that directly predicted the response of a voltage-gated K+ channel within a phospholipid bilayer membrane to applied transmembrane voltages by utilizing very long trajectories was found to be in agreement with the two key experimental results provided by the time-resolved neutron interferometry experiments.

Molecular Mechanism of Aggregation of the Cataract-Related γD-Crystallin W42R Variant from Multiscale Atomistic Simulations.

The mechanisms leading to aggregation of the crystallin proteins of the eye lens remain largely unknown. We use atomistic multiscale molecular simulations to model the solution-state conformational dynamics of γD-crystallin and its cataract-related W42R variant at both infinite dilution and physiologically relevant concentrations. We find that the W42R variant assumes a distinct conformation in solution that leaves the Greek key domains of the native fold largely unaltered but lacks the hydrophobic interdomain interface that is key to the stability of wild-type γD-crystallin. At physiologically relevant concentrations, exposed hydrophobic regions in this alternative conformation become primary sites for enhanced interprotein interactions leading to large-scale aggregation.

Calmodulin Gates Aquaporin 0 Permeability through a Positively Charged Cytoplasmic Loop.

Aquaporin 0 (AQP0), the major intrinsic protein of the eye lens, plays a vital role in maintaining lens clarity by facilitating the transport of water across lens fiber cell membranes. AQP0 reduces its osmotic water permeability constant (Pf) in response to increases in the external calcium concentration, an effect that is mediated by an interaction with the calcium-binding messenger protein, calmodulin (CaM), and phosphorylation of the CaM-binding site abolishes calcium sensitivity. Despite recent structural characterization of the AQP0-CaM complex, the mechanism by which CaM modulates AQP0 remains poorly understood. By combining atomistic molecular dynamics simulations and oocyte permeability assays, we conclude that serine phosphorylation of AQP0 does not inhibit CaM binding to the whole AQP0 protein. Instead, AQP0 phosphorylation alters calcium sensitivity by modifying the AQP0-CaM interaction interface, particularly at an arginine-rich loop that connects the fourth and fifth transmembrane helices. This previously unexplored loop, which sits outside of the canonical CaM-binding site on the AQP0 cytosolic face, mechanically couples CaM to the pore-gating residues of the second constriction site. We show that this allosteric loop is vital for CaM regulation of the channels, facilitating cooperativity between adjacent subunits and regulating factors such as serine phosphorylation. Similar allosteric interactions may also mediate CaM modulation of the properties of other CaM-regulated proteins.

Refining Protein Penetration into the Lipid Bilayer Using Fluorescence Quenching and Molecular Dynamics Simulations: The Case of Diphtheria Toxin Translocation Domain.

Dynamic disorder of the lipid bilayer presents a challenge for establishing structure-function relationships in membranous systems. The resulting structural heterogeneity is especially evident for peripheral and spontaneously inserting membrane proteins, which are not constrained by the well-defined transmembrane topology and exert their action in the context of intimate interaction with lipids. Here, we propose a concerted approach combining depth-dependent fluorescence quenching with Molecular Dynamics simulation to decipher dynamic interactions of membrane proteins with the lipid bilayers. We apply this approach to characterize membrane-mediated action of the diphtheria toxin translocation domain. First, we use a combination of the steady-state and time-resolved fluorescence spectroscopy to characterize bilayer penetration of the NBD probe selectively attached to different sites of the protein into membranes containing lipid-attached nitroxyl quenching groups. The constructed quenching profiles are analyzed with the Distribution Analysis methodology allowing for accurate determination of transverse distribution of the probe. The results obtained for 12 NBD-labeled single-Cys mutants are consistent with the so-called Open-Channel topology model. The experimentally determined quenching profiles for labeling sites corresponding to L350, N373, and P378 were used as initial constraints for positioning TH8-9 hairpin into the lipid bilayer for Molecular Dynamics simulation. Finally, we used alchemical free energy calculations to characterize protonation of E362 in soluble translocation domain and membrane-inserted conformation of its TH8-9 fragment. Our results indicate that membrane partitioning of the neutral E362 is more favorable energetically (by ~ 6 kcal/mol), but causes stronger perturbation of the bilayer, than the charged E362.

Preface: Special Topic on Ions in Water.

This special topic contains a diverse collection of 40 articles that span the vast range of subjects that fall under the heading "Ions in Water," a longstanding mainstay of chemical physics. The investigations reported herein employ state-of-the-art theoretical, computational, and experimental techniques, as well as combinations thereof, to provide new insights into the fundamental aspects of ion solvation and the important roles that ions play in mediating physicochemical processes occurring in solutions and at interfaces in a wide variety of settings relevant to biological, environmental, and technological applications.

Anomalous behavior of water inside the SecY translocon.

The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359-E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a "primed" open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182-17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications.