Secondary relaxations in supercooled and glassy sucrose-borate aqueous solutions

The dielectric relaxation spectra of concentrated aqueous solutions of sucrose-borate mixtures have been measured in the supercooled and glassy regions in the frequency range of 40Hz to 2MHz. The secondary (beta) relaxation process was analyzed in the temperature range 183-233K at water contents between 20 and 30wt%. The relaxation times were obtained, and the activation energy of that process was calculated. In order to assess the effect of borate on the relaxation of disaccharide-water mixtures, we also studied the dielectric behavior of sucrose aqueous solutions in the same range of temperatures and water contents. Our findings support the view that, beyond a water content of approximately 20wt%, the secondary relaxation of water-sucrose and water-sucrose-borate mixtures adopts a universal character that can be explained in terms of a simple exponential function of the temperature scaled by the glass transition temperature (T(g)). The behavior observed for water-sucrose and water-sucrose-borate mixtures is compared with previous results obtained in other water-carbohydrate systems.     

Solvent-Dependent Gating Motions of an Extremophilic Lipase from Pseudomonas aeruginosa

Understanding how organic solvent-stable proteins can function in anhydrous and often complex solutions is essential for the study of the interaction of protein and molecular immiscible interfaces and the design of efficient industrial enzymes in nonaqueous solvents. Using an extremophilic lipase from Pseudomonas aeruginosa as an example, we investigated the conformational dynamics of an organic solvent-tolerant enzyme in complex solvent milieux. Four 100-ns molecular dynamics simulations of the lipase were performed in solvent systems: water, hexane, and two mixtures of hexane and water, 5% and 95% (w/w) hexane. Our results show a solvent-dependent structural change of the protein, especially in the region that regulates the admission of the substrate. We observed that the lipase is much less flexible in hexane than in aqueous solution or at the immiscible interface. Quantified by the size of the accessible channel, the lipase in water has a closed-gate conformation and no access to the active site, while in the hexane-containing systems, the lipase is at various degrees of open-gate state, with the immiscible interface setup being in the widely open conformation ensembles. The composition of explicit solvents in the access channel showed a significant influence on the conformational dynamics of the protein. Interestingly, the slowest step (bottleneck) of the hexane-induced conformational switch seems to be correlated with the slow dehydration dynamics of the channel.     

Dynamical Phase Transitions Reveal Amyloid-like States on Protein Folding Landscapes

Developing an understanding of protein misfolding processes presents a crucial challenge for unlocking the mysteries of human disease. In this article, we present our observations of β-sheet-rich misfolded states on a number of protein dynamical landscapes investigated through molecular dynamics simulation and Markov state models. We employ a nonequilibrium statistical mechanical theory to identify the glassy states in a protein’s dynamics, and we discuss the nonnative, β-sheet-rich states that play a distinct role in the slowest dynamics within seven protein folding systems. We highlight the fundamental similarity between these states and the amyloid structures responsible for many neurodegenerative diseases, and we discuss potential consequences for mechanisms of protein aggregation and intermolecular amyloid formation.     

Dynamical Phase Transitions Reveal Amyloid-like States on Protein Folding Landscapes

Developing an understanding of protein misfolding processes presents a crucial challenge for unlocking the mysteries of human disease. In this article, we present our observations of β-sheet-rich misfolded states on a number of protein dynamical landscapes investigated through molecular dynamics simulation and Markov state models. We employ a nonequilibrium statistical mechanical theory to identify the glassy states in a protein’s dynamics, and we discuss the nonnative, β-sheet-rich states that play a distinct role in the slowest dynamics within seven protein folding systems. We highlight the fundamental similarity between these states and the amyloid structures responsible for many neurodegenerative diseases, and we discuss potential consequences for mechanisms of protein aggregation and intermolecular amyloid formation.     

Comparison of the Conformational Behavior of Sialyllactose Complexed with the two Viral Attachment Proteins Influenza A Hemagglutinin and the Murine Polyomavirus

The attachment of a virus to the host cell surface is influenced by enthalpic and entropic factors. A detailed evaluation of all possible energetic interactions including the effects of solvent molecules seems to be a promising way to gain deeper insights in the overall process of binding. Here we performed intensive molecular dynamics studies to compare the conformational space available for the unbound sialyllactose in aqueous solution and when complexed with influenza A hemagglutinin and the murine polyoma virus. In general the conformational freedom of sialyllactose is considerably reduced compared to the free state. Remarkably, two different conformations of the Siaα(2-3)Galβ glycosidic linkages are preferred (which are both populated in the free state) when complexed with either protein.     

Controlling the protein dynamical transition with sugar-based bioprotectant matrices: a neutron scattering study

Through elastic neutron scattering we measured the mean-square displacements of the hydrogen atoms of lysozyme embedded in a glucose-water glassy matrix as a function of the temperature and at various water contents. The elastic intensity of all the samples has been interpreted in terms of the double-well model in the whole temperature range. The dry sample shows an onset of anharmonicity at approximately 100 K, which can be attributed to the activation of methyl group reorientations. Such a protein intrinsic dynamics is decoupled from the external environment on the whole investigated temperature range. In the hydrated samples an additional and larger anharmonic contribution is provided by the protein dynamical transition, which appears at a higher temperature Td. As hydration increases the coupling between the protein internal dynamics and the surrounding matrix relaxations becomes more effective. The behavior of Td that, as a function of the water content, diminishes by approximately 60 K, supports the picture of the protein dynamics as driven by solvent relaxations. A possible connection between the protein dynamical response versus T and the thermal stability in glucose-water bioprotectant matrices is proposed.     

Control of electron transfer by protein dynamics in photosynthetic reaction centers

Abstract

Trehalose and glycerol are low molecular mass sugars/polyols that have found widespread use in the protection of native protein states, in both short- and long-term storage of biological materials, and as a means of understanding protein dynamics. These myriad uses are often attributed to their ability to form an amorphous glassy matrix. In glycerol, the glass is formed only at cryogenic temperatures, while in trehalose, the glass is formed at room temperature, but only upon dehydration of the sample. While much work has been carried out to elucidate a mechanistic view of how each of these matrices interact with proteins to provide stability, rarely have the effects of these two independent systems been directly compared to each other. This review aims to compile decades of research on how different glassy matrices affect two types of photosynthetic proteins: (i) the Type II bacterial reaction center from Rhodobacter sphaeroides and (ii) the Type I Photosystem I reaction center from cyanobacteria. By comparing aggregate data on electron transfer, protein structure, and protein dynamics, it appears that the effects of these two distinct matrices are remarkably similar. Both seem to cause a “tightening” of the solvation shell when in a glassy state, resulting in severely restricted conformational mobility of the protein and associated water molecules. Thus, trehalose appears to be able to mimic, at room temperature, nearly all of the effects on protein dynamics observed in low temperature glycerol glasses.     

Nitrogen and Phosphorus Release from Mixed Litter Layers is Lower than Predicted from Single Species Decay

Ecosystem-level nutrient dynamics during decomposition are often estimated from litter monocultures. If species effects are additive, we can statistically predict nutrient dynamics in multi-species systems from monoculture work, and potential consequences of species loss. However, if species effects are dependent on interactions with other litter species (that is, non-additive), predictions based on monoculture data will likely be inaccurate. We conducted a 3-year, full-factorial, mixed-litter decomposition study of four dominant tree species in a temperate forest and measured nitrogen and phosphorus dynamics to explore whether nutrient dynamics in mixtures were additive or non-additive. Following common approaches, we used litterfall data to predict nutrient dynamics at the ecosystem-level. In mixtures, we observed non-additive effects of litter mixing on nutrient dynamics: the presence of nutrient-rich species in mixture facilitated nutrient release, whereas nutrient-poor species facilitated nutrient retention. Fewer nutrients were released from mixtures containing high-quality litter, and more immobilized from mixtures containing low-quality litter, than predicted from monocultures, creating a difference in overall nutrient release between predicted and actual dynamics in litter mixtures. Nutrient release at the ecosystem-level was greatly overestimated when based on monocultures because the effect of species interactions on nutrient immobilization was not accounted for. Our data illustrate that the identity of species in mixtures is key to their role in non-additive interactions, with repercussions for mineral nutrient availability and storage. These results suggest that predictions of ecosystem-level nutrient dynamics using litter monoculture data likely do not accurately represent actual dynamics because the effects of litter species interactions are not incorporated. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dielectric spectroscopy study of myoglobin in glycerol-water mixtures

Due to the interest in protein dynamics, there are numerous dielectric relaxation studies of proteins in water and in glass-forming aqueous solvents such as glycerol-water mixtures. In the regime of low frequencies, the inevitable dc-conductivity of such systems limits the resolution of dynamics that are slow compared with the solvent relaxation. Solutions of myoglobin in glycerol/water mixtures of various compositions are measured by dielectric spectroscopy in the frequency range from 10 mHz to 10 MHz. The resolution of low frequency modes is improved by two approaches: electrical ‘cleaning’ and the analysis of the derivative of the real component of permittivity, which shows no direct signature of dc-conductivity. Effects of internal interfacial polarization are also addressed by measuring the same solvents in confinement as well as mixed with glass beads. We find two processes, the structural relaxation of the solvent and the slower rotational mode of the protein, with no indication at even lower frequencies of a dielectric signature of fluctuations associated with protein dynamics.     

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer‐membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co‐occur and are from neighboring gene in Salmonella Typhi‐occurrence of homologs of both STY3178 and STY3179 are identified in many Gram‐negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra‐cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra‐cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin.     

Solvent-induced ligand dissociation and conformational states of Cellular Retinol-Binding Protein type I

Cellular Retinol-Binding Protein type I (CRBP) exhibits very high affinity for its ligand, bound within a buried cavity completely shielded from the outside medium. Three-dimensional structure and backbone dynamics in aqueous solution at neutral pH, either in the absence or in the presence of retinol, fail to represent the protein in a state capable of ligand uptake and release. The question was asked whether changes in the composition of the outside medium might facilitate ligand dissociation. Acidic aqueous solutions and water-alcohol mixtures were selected, among the best described denaturing solvents, to investigate their effects on the stability of the carrier-ligand complex and the conformational state of the protein upon ligand release. Circular dichroism (CD) and fluorescence spectroscopy were used to probe protein secondary and tertiary structure, compactness and retinol dissociation. While in purely aqueous media retinol dissociation parallels the acid-induced denaturation of the carrier, in water-alcohol mixtures it occurs in a range of co-solvent content lower than that required for protein denaturation. In light of these results, it is suggested that local solvent properties in vivo might modulate protein conformation and flexibility and thus play a fundamental role in the control of retinol exchange between carrier and membrane-bound donors and acceptors.     

Dehydration-induced conformational changes of poly-l-lysine as influenced by drying rate and carbohydrates

The conformation of hydrated and air-dried poly-l-lysine in thin films was studied using Fourier transform IR spectroscopy in the amide-I region. Hydrated poly-l-lysine has a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, an extended β-sheet conformation is adopted. This conformational transition can be prevented by fast air-drying within 2–3 min. Slow air-drying in the presence of sucrose also preserves the aqueous conformation and results in the formation of a glassy state. Comparison of shifts of the OH band with temperature indicates that sucrose/poly-l-lysine mixtures form a molecularly more densely packed glassy matrix, having a higher glass transition temperature (T_g), than sucrose alone. Whether direct interaction of sugar and polypeptide or glass formation is involved in the stabilization during slow air-drying was studied by drying in the presence of glucose or dextran. Compared with dextran (and sucrose to a lesser extent), glucose gives superior protection. Dried glucose has the lowest T_g and the best interacting properties. We conclude that either immobilization by fast air-drying or sufficient interaction with a protectant through hydrogen bonding (slow drying) plays the leading role in the preservation of the aqueous protein structure.     

Sorption of water by bidisperse mixtures of carbohydrates in glassy and rubbery states

Water sorption by bidisperse carbohydrate mixtures consisting of varying ratios of a narrow-molecular-weight distribution maltopolymer and the disaccharide maltose is investigated to establish a quantitative relation between the composition of the carbohydrate system and the water sorption isotherm. The sorption of water is approached from two limiting cases: the glassy state at low water content and the dilute aqueous carbohydrate solution. In the glassy state, the water content at a given water activity decreases with increasing maltose content of the matrix, whereas in the rubbery state it increases with increasing maltose content. The water sorption behavior in the glassy state is quantified using a variety of models, including the often-utilized but physically poorly founded Guggenheim-Anderson-de Boer model, several variants of the free-volume theory of sorption by glassy polymers, and a two-state sorption model introduced in the present paper. It is demonstrated that both the free-volume models and the two-state sorption model, which all encompass the Flory-Huggins theory for the rubbery-state sorption but which differ in their modeling of the glassy-state sorption, provide a physically consistent foundation for the analysis of water sorption by the carbohydrate matrixes.     

Molecular dynamics simulations of microstructure and mixing dynamics of cryoprotective solvents in water and in the presence of a lipid membrane

Molecular dynamics (MD) simulation is used to investigate the solubility behavior of cryoprotective (CP) solvents, such as DMSO, ethylene glycol (EG) and glycerol (GL), in pure water and in the presence of a lipid membrane. The MD study is focused on an equilibration timescale required for mixing large CP aggregates with aqueous and aqueous/lipid environments. The MD analysis demonstrates that DMSO mixes rapidly with water, so that all solute molecules are uniformly distributed in the equilibrium aqueous solution. Our investigation of the microstructure of binary EG/water and GL/water systems reveals that, despite the miscibility of both CP solvents with water, they are not ideally mixed in aqueous solutions at the molecular level. The MD simulations show that the mixing dynamics of the large CP cluster and surrounding water is found to be strongly dependent on nature of hydrophilic and hydrophobic interactions acting between cryoprotectant molecules. In particular, a spatial hydrogen-bond network formed between CP molecules plays an important role in the mixing dynamics between CP agents and water. A further analysis on the mixing behavior of the CP solvents with pure water and with aqueous solutions at a lipid membrane interface shows that, due to strong binding of the CP molecules to membrane surface, the equilibration process in the lipid environment becomes very slow, at least of the order of microseconds. The MD results are discussed in the context of the better understanding on the composition of the aqueous mixtures of the EG and GL solvents. Knowledge of the microstructure and the dynamics of these systems helps to develop better cryopreservation protocols and to propose more optimal cooling/warming regimes for cellular cryosolutions.     

Electrophysiological responses of taste cells to nutrient mixtures in the polyphagous caterpillar of Grammia geneura

In a normally feeding insect, the taste receptors are exposed to complex mixtures of chemicals, not single compounds. We investigate the responses of neurons in the galeal sensilla of the caterpillar of Grammia geneura to mixtures of nutrient compounds at concentrations occurring in plants. Compounds that stimulated the same neuron were generally additive in their effects in binary mixtures. Amino acids that did not stimulate usually had no effect in mixtures with a stimulating compound, but glutamic acid reduced the response to serine in the medial sensillum. Nutrient compounds that stimulated different cells in a sensillum acted independently of each other. Complex mixtures of amino acids resembling samples of free amino acids from three host plants were less stimulating than expected from their molar concentrations. In host plant selection, the response from the medial sensillum is probably dominated by sucrose; unless sucrose levels are low, amino acids will contribute little to sensory input because they stimulate the same cell as sucrose. In the lateral sensillum, amino acids act independently of sugars. The limited contact chemosensory array of caterpillars seems inadequate to allow them to make fine distinctions between plants on the basis of their free amino acids.     

Time-resolved fluorescence and circular dichroism of porphyrin cytochrome c and Zn-porphyrin cytochrome c incorporated in reversed micelles

Interactions between fluorescent horse heart cytochrome c derivatives (e. g. porphyrin cytochrome c and Zn-porphyrin cytochrome c) with surfactant interfaces in reversed micellar solutions have been studied, using different spectroscopic techniques. Anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] and cationic (cetyltrime-thylammonium bromide, CTAB) surfactant solutions have been used in order to investigate the effects of charge interactions between proteins and interfaces. Circular dichroism reveals that much of the protein secondary structure is lost in AOT-reversed micelles, especially when the molar water/surfactant ratio, wo, is high (wo = 40), whereas in CTAB-reversed micelles secondary structure seems to be preserved. Time-resolved fluorescence measurements of the porphyrin in the cytochrome c molecule yields information about the changes in structure and the dynamics of the protein upon interaction with surfactant assemblies both in aqueous and in hydrocarbon solutions. With AOT as surfactant a strong interaction between protein and interface can be observed. The effects found in aqueous AOT solution are of the same kind as in hydrocarbon solution. In the CTAB systems the interactions between protein and surfactant are much less pronounced. The measured effects on the fluorescence properties of the proteins are different in aqueous and hydrocarbon solutions. In general, the observations can be explained by an electrostatic attraction between the overall positively charged protein molecules and the anionic AOT interface. Electrostatic attraction can also occur between the cytochrome c derivatives and CTAB because there is a negatively charged zone on the surface of the proteins. From the fluorescence anisotropy decays it can be concluded that in the CTAB-reversed micellar system these interactions are not important, whereas in an aqueous CTAB solution the proteins interact with surfactant molecules.     

Relaxation dynamics of a protein solution investigated by dielectric spectroscopy

In the present work, we provide a dielectric study on two differently concentrated aqueous lysozyme solutions in the frequency range from 1MHz to 40GHz and for temperatures from 275 to 330K. We analyze the three dispersion regions, commonly found in protein solutions, usually termed β-, γ-, and δ-relaxations. The β-relaxation, occurring in the frequency range around 10MHz and the γ-relaxation around 20GHz (at room temperature) can be attributed to the rotation of the polar protein molecules in their aqueous medium and the reorientational motion of the free water molecules, respectively. The nature of the δ-relaxation, which is often ascribed to the motion of bound water molecules, is not yet fully understood. Here we provide data on the temperature dependence of the relaxation times and relaxation strengths of all three detected processes and on the dc conductivity arising from ionic charge transport. The temperature dependences of the β- and γ-relaxations are closely correlated. We found a significant temperature dependence of the dipole moment of the protein, indicating conformational changes. Moreover we find a breakdown of the Debye-Stokes-Einstein relation in this protein solution, i.e., the dc conductivity is not completely governed by the mobility of the solvent molecules. Instead it seems that the dc conductivity is closely connected to the hydration shell dynamics.     

A method for three-dimensional protein characterization and its application to a complex plant (corn) extract

Knowledge of host protein properties is critical for developing purification methods for recombinant proteins from a specific host, or for choosing suitable hosts and targeted expression tissues for a specific recombinant protein. A method to obtain a three-dimensional (3D) map (surface hydrophobicity (SH), isoelectric point (pI), and molecular weight (MW)), of a host's aqueous soluble protein properties was developed. The method consists of hydrophobic partitioning in a PEG 3350 (15.7%)-Na(2)SO(4) (8.9%)-NaCl (3%) aqueous two-phase (ATP) system followed by quantitative, 2D-electrophoretic characterization of the proteins of each equilibrium phase and the original extract. The pI and MW of host proteins were obtained directly through 2D electrophoresis. The partition coefficients of individual proteins were obtained by quantitative matching of protein spots in the top and bottom phase gels and calculating the protein partition coefficients from this information. Correlation of the partition coefficient to a SH scale was established by partitioning several model proteins with known surface hydrophobicities in the same ATP system. The inclusion of the extract gel provided for a spot selection criterion based on satisfactory mass balance closure. The method is illustrated by application to a mixture of model proteins and to complex mixtures, that is, corn germ proteins extracted at pH 7 and pH 4.     

Metacommunity of a host metapopulation: explaining patterns and structures of a fish parasite metacommunity in a Neotropical floodplain basin

Host-parasite metacommunities are influenced by a myriad of factors, although little is known about which processes affect this relationship at different scales. Here, we tested how local habitat characteristics and host traits explained the parasite metacommunity of a migratory fish in a large Brazilian river floodplain. The parasite metacommunity structure showed a Clementsian pattern, which indicates a more deterministic assembly pattern, in accordance with partial Redundancy Analysis results. Results indicated that species filtering is the predominant mechanism driving community assembly. Patterns were clearer in the dry season of the floodplain. Environmental determinism seems to explain ectoparasite metacommunities in the dry season, in contrast with endoparasites that were more correlated to host traits. Overall, our results indicated that ectoparasitism is an interaction marked by opportunity, whereas endoparasitism is likely related to host features. Thus, we argue that metacommunity structuring of parasites depends on the infection strategy. Our results show that floodplain dynamics are central not only for free-living animal organizations but also for symbiotic interactions. Here, we highlight the importance of understanding the factors influencing the distribution of parasites to predict their transmission, as well as the importance of floodplain dynamics and its hydrological regime on the maintenance of ecological interactions.