Proteins can be denatured by pressures of a few hundred MPa. This finding apparently contradicts the most widely used model of protein stability, where the formation of a hydrophobic core drives protein folding. The pressure denaturation puzzle is resolved by focusing on the pressure-dependent transfer of water into the protein interior, in contrast to the transfer of nonpolar residues into water, the approach commonly taken in models of protein unfolding. Pressure denaturation of proteins can then be explained by the pressure destabilization of hydrophobic aggregates by using an information theory model of hydrophobic interactions. Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure with water molecules penetrating their core. Activation volumes for hydrophobic contributions to protein folding and unfolding kinetics are positive. Clathrate hydrates are predicted to form by virtually the same mechanism that drives pressure denaturation of proteins.A decade ago, Walter Kauzmann (1) challenged the commonly held view that a hydrophobic core stabilizes globular proteins, by poignantly remarking that the ''liquid-hydrocarbon model (2) fails almost completely when one attempts to extend it to the effects of pressure on protein folding.'' Although a variety of forces stabilize folded proteins (3-6), the formation of a hydrophobic core is thought to play a dominant role. This view is supported by the temperature dependence of hydrophobic contributions to protein unfolding showing remarkable similarities to the transfer of hydrocarbons from a nonpolar phase into water, notably a convergence of the entropy of transfer (2,7,8). However, Kauzmann (1) pointed out that the pressure dependence of protein unfolding is at odds with the hydrophobic-core model: The volume change ⌬V upon unfolding is positive at low pressures but negative at pressures of about 100-200 MPa. The transfer of hydrocarbons into water shows exactly the opposite behavior, with ⌬V being negative at low pressures and positive at high pressures.Evidently, pressure unfolding of a protein (9-16) does not correspond to the transfer of a nonpolar molecule from a nonpolar environment into aqueous solution. Unlike heatdenatured proteins, the ensemble of pressure-denatured proteins retains elements of structural organization (13, 17). Consequently, an understanding of the thermodynamics of pressure denaturation might focus on the free energy of water transfer into the hydrophobic core of the protein (18) rather than transfer of nonpolar solutes into water. Our conceptual framework for pressure denaturation is as follows: the protein interior is largely composed of efficiently packed residues, more likely hydrophobic than those at the surface (19). Increasing hydrostatic pressure then forces water molecules into the protein interior, gradually filling cavities, and eventually breaking the protein structure apart.We therefore study the effects of pressure on the association of nonpolar residues in water. We use the informat...

A theoretical approach is developed to quantify hydrophobic hydration and interactions on a molecular scale, with the goal of insight into the molecular origins of hydrophobic effects. The model is based on the fundamental relation between the probability for cavity formation in bulk water resulting from molecular-scale density fluctuations, and the hydration free energy of the simplest hydrophobic solutes, hard particles. This probability is estimated using an information theory (IT) approach, incorporating experimentally available properties of bulk water -the density and radial distribution function. The IT approach reproduces the simplest hydrophobic effects: hydration of spherical nonpolar solutes, the potential of mean force (PMF) between methane molecules, and solvent contributions to the torsional equilibrium of butane. Applications of this approach to study temperature and pressure effects provide new insights into the thermodynamics and kinetics of protein folding. The IT model relates the hydrophobic-entropy convergence observed in protein unfolding experiments to the macroscopic isothermal compressibility of water. A novel explanation for pressure denaturation of proteins follows from an analysis of the pressure stability of hydrophobic aggregates, suggesting that water penetrates the hydrophobic core of proteins at high pressures. This resolves a long-standing puzzle, whether pressure denaturation contradicts the hydrophobic-core model of protein stability. Finally, issues of "dewetting" of molecularly large nonpolar solutes are discussed in the context of a recently developed perturbation theory approach.

SynopsisExperimental data on gas sorption and polymer swelling in glassy polymer-gas systems a t elevated pressures are presented for CO, with polycarbonate, poly(methy1 methacrylate), and polystyrene over a range of temperatures from 33 to 65°C and pressures up to 100 atm. The swelling and sorption behavior were found to depend on the occurrence of a glass transition for the polymer induced by the sorption of CO,. Two distinct types of swelling and sorption isotherms were measured. One isotherm is characterized by swelling and sorption that reach limiting values a t elevated pressures. The other isotherm is characterized by swelling and sorption that continue to increase with pressure and a pressure effect on swelling that is somewhat greater than the effect of pressure on sorption. Glass transition pressures estimated from the experimental results for polystyrene with CO, are used to obtain the relationship between CO, solubility and the g l a s transition temperature for the polymer. This relationship is in very good agreement with a theoretical corresponding-states correlation for glass transition temperatures of polystyrene-liquid diluent mixtures.

• Macrophage-derived microvesicles induced cellular differentiation in naive monocytes.• Macrophage-derived microvesicles shuttle of miRNAs to target cells.Microvesicles are small membrane-bound particles comprised of exosomes and varioussized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. (Blood. 2013;121(6):984-995)

An information theory model is used to construct a molecular explanation why hydrophobic solvation entropies measured in calorimetry of protein unfolding converge at a common temperature. The entropy convergence follows from the weak temperature dependence of occupancy fluctuations for molecular-scale volumes in water. The macroscopic expression of the contrasting entropic behavior between water and common organic solvents is the relative temperature insensitivity of the water isothermal compressibility. The information theory model provides a quantitative description of small molecule hydration and predicts a negative entropy at convergence. Interpretations of entropic contributions to protein folding should account for this result. 87.15.Da, 87.15.Kg High sensitivity calorimetry on the unfolding of globular proteins has suggested that hydrophobic contributions to the entropies of unfolding converge to zero near 385 K [1][2][3][4][5]. Additional thermodynamic information on protein folding processes is then obtained by extrapolating the calorimetric measurements to the convergence temperature where hydrophobic contributions vanish. The convergence behavior of entropies of solution of hydrocarbons in water is known also [6][7][8][9]]. Yet a microscopic-level mechanism for this phenomenon has not been offered [10]. Here we identify a mechanism by analysis of an information theory model of hydrophobic hydration [11,12]. We show how the theory predicts entropy convergence on the basis of the density and density fluctuations of liquid water. Consistent with experimental results on hydrophobic hydration, the model predicts that the entropy at convergence should be negative.Aqueous protein solutions are complex systems involving molecular interactions of several kinds, including those associated with ionic and polar groups, in addition to hydrophobic contributions [5]. For clarity we focus on strictly hydrophobic species and idealize those solutes as hard core objects that perfectly repel water molecule centers identified as the position of the oxygen atoms. Statistical mechanics relates the excess chemical potential of hard core solutes to the probability, p 0 , of finding an empty volume, v, or a cavity of a given size and shape in water,We calculate p 0 by considering the probabilities, p n , of observing exactly n solvent centers in the cavity. The p n are predicted by maximizing an information entropy [11,12], subject to the constraints of available information. The experimentally accessible first and second moments of the number of solvent centers in the cavity region constitute the generally available information. This procedure yields here the distribution p n = exp(λ 0 + λ 1 n + λ 2 n 2 ) where λ 0 , λ 1 , and λ 2 are Lagrange multipliers. The required moments are obtained from the number density ρ and oxygen-oxygen radial distribution function g(r) byThat this is an accurate model for the circumstances considered here has been explicitly verified [11,12]. Figure 1 shows the calculated ∆µ ex for spherical ...

An understanding of molecular statistical thermodynamic theory is fundamental to the appreciation of molecular solutions. This complex subject has been simplified by the authors with down-to-earth presentations of molecular theory. Using the potential distribution theorem (PDT) as the basis, the text provides an up-to-date discussion of practical theories in conjunction with simulation results. The authors discuss the field in a concise and simple manner, illustrating the text with useful models of solution thermodynamics and numerous exercises. Modern quasi-chemical theories that permit statistical thermodynamic properties to be studied on the basis of electronic structure calculations are given extended development, as is the testing of those theoretical results with ab initio molecular dynamics simulations. The book is intended for students undertaking research problems of molecular science in chemistry, chemical engineering, biochemistry, pharmaceutical chemistry, nanotechnology, and biotechnology.

Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up-regulation in fibrotic or steatotic livers, or in culture-activated or ethanol-treated primary mouse HSC is associated with a reciprocal down-regulation of microRNA-214 (miR-214). By using protector or reporter assays to investigate the 3′-untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis-inducing stimuli was due to reduced expression of miR-214 which otherwise inhibited CCN2 expression by directly binding to the CCN2 3′-UTR. Additionally, miR-214 was present in HSC exosomes, which were bi-membrane vesicles, 50–150nm in diameter, negatively charged (−26mV), and positive for CD9. MiR-214 levels in exosomes but not in cell lysates were reduced by pre-treatment of the cells with the exosome inhibitor, GW4869. Co-culture of miR-214-transfected donor HSC with CCN2 3′-UTR luciferase reporter-transfected recipient HSC resulted in miR-214- and exosome-dependent regulation of a wild type CCN2 3′-UTR reporter but not of a mutant CCN2 3′-UTR reporter lacking the miR-214 binding site. Exosomes from HSC were a conduit for uptake of miR-214 by primary mouse hepatocytes. Down-regulation of CCN2 expression by miR-214 also occurred in human LX-2 HSC, consistent with a conserved miR-214 binding site in the human CCN2 3′-UTR. MiR-214 in LX-2 cells was shuttled via exosomes to recipient LX-2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3′-UTR activity or expression of CCN2 downstream targets, including αSMA or collagen. Experimental fibrosis in mice was associated with reduced circulating miR-214 levels. Conclusion Exosomal transfer of miR-214 is a paradigm for the regulation of CCN2-dependent fibrogenesis and identifies fibrotic pathways as targets of epigenetic regulation by exosomal miRs.

Using Monte Carlo simulations, we investigated the influence of solute size and solute-water attractive interactions on hydration water structure around spherical clusters of 1, 13, 57, 135, and 305 hexagonally close-packed methanes and the single hard-sphere (HS) solute analogues of these clusters. We obtain quantitative results on the density of water molecules in contact with the HS solutes as a function of solute size for HS radii between 3.25 and 16.45 A. Analysis of these results based on scaled-particle theory yields a hydration free energy/surface area coefficient equal to 139 cal/(mol A2), independent of solute size, when this coefficient is defined with respect to the van der Waals surface of the solute. The same coefficient defined with respect to the solvent-accessible surface decreases with decreasing solute size for HS radii less than approximately 10 A. We also find that solute-water attractive interactions play an important role in the hydration of the methane clusters. Water densities in the first hydration shell of the three largest clusters are greater than bulk water density and are insensitive to the cluster size. In contrast, contact water densities for the HS analogues of these clusters decrease with solute size, falling below the bulk density of water for the two largest solutes. Thus, the large HS solutes dewet, while methane clusters of the same size do not.