Peptide conformational preferences in osmolyte solutions: transfer free energies of decaalanine

The nature in which the protecting osmolyte trimethylamine N-oxide (TMAO) and the denaturing osmolyte urea affect protein stability is investigated, simulating a decaalanine peptide model in multiple conformations of the denatured ensemble. Binary solutions of both osmolytes and mixed osmolyte solutions at physiologically relevant concentrations of 2:1 (urea:TMAO) are studied using standard molecular dynamics simulations and solvation free energy calculations. Component analysis reveals the differences in the importance of the van der Waals (vdW) and electrostatic interactions for protecting and denaturing osmolytes. We find that urea denaturation governed by transfer free energy differences is dominated by vdW attractions, whereas TMAO exerts its effect by causing unfavorable electrostatic interactions both in the binary solution and mixed osmolyte solution. Analysis of the results showed no evidence in the ternary solution of disruption of the correlations among the peptide and osmolytes, nor of significant changes in the strength of the water hydrogen bond network.     

Hydrophobic interactions in presence of osmolytes urea and trimethylamine-N-oxide

Molecular dynamics simulations were carried out to study the influences of two naturally occurring osmolytes, urea, and trimethylamine-N-oxide (TMAO) on the hydrophobic interactions between neopentane molecules. In this study, we used two different models of neopentane: One is of single united site (UA) and another contains five-sites. We observe that, these two neopentane models behave differently in pure water as well as solutions containing osmolytes. Presence of urea molecules increases the stability of solvent-separated state for five-site model, whereas osmolytes have negligible effect in regard to clustering of UA model of neopentane. For both models, dehydration of neopentane and preferential solvation of it by urea and TMAO over water molecules are also observed. We also find the collapse of the second-shell of water by urea and water structure enhancement by TMAO. The orientational distributions of water molecules around different layers of neopentane were also calculated and we find that orientation of water molecules near to hydrophobic moiety is anisotropic and osmolytes have negligible effect on it. We also observe osmolyte-induced water-water hydrogen bond life time increase in the hydration shell of neopentane as well as in the subsequent water layers.     

Structure of Aqueous Solutions of Trimethylaminoxide,Urea, and Their Mixture

Aqueous solutions of natural osmolytes (trimethylaminoxide (TMAO), urea, and their mixture) at relatively small (biologically relevant) concentrations are analyzed by the all-atom molecular dynamics simulation. In the recent work (Smolin N. et al. PCCP. 2017. 19. P. 6345) it has been noted that in the protein hydration shell the fraction of TMAO molecules is much smaller than that of urea. The urea addition causes a further decrease in the TMAO fraction in the protein hydration shell. This work shows that in binary solutions urea fraction at urea molecules is always larger than the bulk urea concentration. At the same time, the TMAO fraction near TMAO is the same as in the bulk. In ternary solutions, TMAO and urea behave the same as the binary ones, i.e. they do not noticeably affect each other. This means that the behavior of TMAO and urea molecules in the protein hydration shell is associated with protein rather than their interaction with each other.     

Trimethylamine N-oxide stabilizes RNA tertiary structure and attenuates the denaturating effects of urea

Trimethylamine N-oxide (TMAO) and urea are osmolytes. Osmolytes allow cells to remain viable in harsh or extreme environments. Both TMAO and urea are found in shark and rays at approximate molar ratios of 1:2, respectively. At this ratio TMAO nearly completely counteracts the destabilizing effects that urea has on proteins. We ask whether RNA, which is denatured by urea, is stabilized by TMAO in a manner similar to that seen for proteins. We found that TMAO stabilizes Escherichia coli tRNAfmet tertiary structure and counteracts the denaturing effects of urea at the same ratios found for proteins. Cation binding usually drives RNA tertiary structure formation. These results suggest that tertiary structure stability is not only sensitive to cations but also to the aqueous composition and properties of the solvent. We propose that tertiary structure folding is driven by unfavorable interactions between TMAO and the phosphodiester backbone.     

Hydrophobic interactions in urea-trimethylamine-N-oxide solutions

The influence of osmolytes urea and trimethylamine- N-oxide (TMAO) on hydrophobic interactions is investigated employing molecular dynamics simulations. These osmolytes are of interest because of their opposing influence on proteins in solution; the denaturing effect of urea can be countered with TMAO. A neopentane pair is used to model typical nonpolar entities. Binary water-urea and water-TMAO as well as ternary water-urea-TMAO systems are considered. Neopentane-neopentane potentials of mean force, neopentane-water, and neopentane-osmolyte distribution functions are reported. Urea is found to have only modest influence on the neopentane-neopentane potential of mean force, but the hydrophobic attraction is completely destroyed by the presence of TMAO. It is shown that TMAO tends to act as a simple "surfactant" displacing water and urea (if it is present) from the first solvation shell of neopentane. It is likely the surfactant-like influence of TMAO that accounts for the elimination of the hydrophobic attraction. The implications of our results for explanations of the action of TMAO in protein solutions are discussed.     

Volume exclusion and H-bonding dominate the thermodynamics and solvation of trimethylamine-N-oxide in aqueous urea

Trimethylamine-N-oxide (TMAO) and urea represent the extremes among the naturally occurring organic osmolytes in terms of their ability to stabilize/destabilize proteins. Their mixtures are found in nature and have generated interest in terms of both their physiological role and their potential use as additives in various applications (crystallography, drug formulation, etc.). Here we report experimental density and activity coefficient data for aqueous mixtures of TMAO with urea. From these data we derive the thermodynamics and solvation properties of the osmolytes, using Kirkwood-Buff theory. Strong hydrogen-bonding at the TMAO oxygen, combined with volume exclusion, accounts for the thermodynamics and solvation of TMAO in aqueous urea. As a result, TMAO behaves in a manner that is surprisingly similar to that of hard-spheres. There are two mandatory solvation sites. In plain water, these sites are occupied with water molecules, which are seamlessly replaced by urea, in proportion to its volume fraction. We discuss how this result gives an explanation both for the exceptionally strong exclusion of TMAO from peptide groups and for the experimentally observed synergy between urea and TMAO.     

The effect of trimethylamine N-oxide on RNase a stability

The thermal stability of bovine pancreatic ribonuclease (RNase A) has been investigated in the presence of trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, by means of differential scanning calorimetry (DSC) and circular dichroism (CD) measurements at neutral and acid pH conditions. It is well known that compatible osmolytes such as TMAO are effective in stabilizing protein structure and counteracting the denaturing the effect of urea and guanidinium hydrochloride (GuHCl). Calorimetric results show that TMAO stabilizes RNase A at pH 7.0 and does not stabilize the protein at pH 4.0. RNase A thermal denaturation in the presence of TMAO is a reversible two-state N ⇆ D process. We also show that TMAO counteracts the urea and GuHCl denaturing effect at neutral pH, whereas the counteracting ability is lost at acid pH.     

Modulation of hydrophobic effect by cosolutes

This work concerns a comparison of the hydration properties and self-association behavior in aqueous solution of three biologically relevant simple molecules: tert-butyl alcohol (TBA), trimethylamine-n-oxide (TMAO), and glycine betaine (GB). These molecules were used as a model to study hydrophobic behavior in water solutions. In particular, water perturbation induced by TBA, TMAO, and GB molecules was studied as a function of the solute molar fraction X(2) (0 < X(2) < 0.04) by Raman spectra of water in the fundamental OH-stretching region (3,800-2,800 cm(-1)). Furthermore, possible hydrophobic clustering of these molecules was investigated by studying the behavior of the alkyl CH stretching band in the 3,100-2,900 cm(-1) frequency region as a function of X(2). To establish the existence of a correlation between the effects of these three solutes on the micellization process and changes in the properties of the solvent, the behavior of the critical micelle concentration of sodium dodecyl sulfate was also investigated as a function of the added amount of TBA, TMAO, and GB. On the whole, these data show that there is no direct correlation between a solute's effect on the water structure and its effect on micelle or protein stability. Results indicate that, while TBA starts to self-aggregate at approximately X(2) = 0.025, both TMAO and GB do not exhibit any significant self-aggregation up to the highest concentration considered. In addition, nonadditive perturbations of the H-bonded networks of solvent water are observed in the case of TBA solutions, but are absent in both the TMAO and GB cases. The absence of these nonlinear effects in TMAO and GB water solutions allow for tracing the microscopical mechanism of the neutrality of these osmolytes toward hydrophobic effects. This confers the compatibility to these two osmolytes, which can be accumulated at high concentrations without interfering with biochemical processes in the cell.     

Effect of Osmolytes on Pressure‐Induced Unfolding of Proteins: A High‐Pressure SAXS Study

Herein, we explore the effect of different types of osmolytes on the high‐pressure stability and tertiary structure of a well‐characterized monomeric protein, staphylococcal nuclease (SNase). Changes in the denaturation pressure and the radius of gyration are obtained in the presence of different concentrations of trimethylamine N‐oxide (TMAO), glycerol and urea. To reveal structural changes in the protein upon compression at various osmolyte conditions, small‐angle X‐ray scattering (SAXS) experiments were carried out. To this end, a new high‐pressure cell suitable for high‐precision SAXS studies at synchrotron sources was built, which allows one to carry out scattering experiments up to maximum pressures of about 7 kbar. Our data clearly indicate that the osmolytes that stabilize proteins against temperature‐induced unfolding drastically increase their pressure stability and that the elliptically shaped curve of the pressure–temperature–stability diagram of proteins is shifted to higher temperatures and pressures with increasing osmolyte concentration. A drastic stabilization is observed for the osmolyte TMAO, which exhibits not only a significant stabilization against temperature‐induced unfolding, but also a particularly strong stabilization of the protein against pressure. In fact, such findings are in accordance with in vivo studies (for example P. J. Yancey, J. Exp. Biol. 2005 , 208, 2819–2830), where unusually high TMAO concentrations in some deep‐sea animals were found. Conversely, chaotropic agents such as urea have a strong destabilizing effect on both the temperature and pressure stability of the protein. Our data also indicate that sufficiently high TMAO concentrations might be able to largely offset the destabilizing effect of urea. The different scenarios observed are discussed in the context of recent experimental and theoretical studies.     

Structure and interaction in aqueous urea-trimethylamine-N-oxide solutions

The structural and energetic properties of solutions containing water, urea, and trimethylamine-N-oxide (TMAO) are examined using molecular dynamics simulations. Such systems are of interest mainly because TMAO acts to counter the protein denaturing effect of urea. Even at relatively high concentration, TMAO is found to fit well into the urea-water structure. The underlying solution structure is influenced by TMAO, but these perturbations tend to be modest. The TMAO-water and TMAO-urea interaction energies make an important contribution to the total energy in solutions where counter-denaturing effects are expected. TMAO-water and TMAO-urea hydrogen bonds have the largest hydrogen-bond energies in the system. Additionally, TMAO cannot hydrogen bond with itself, and hence it interacts strongly with water and urea. These observations suggest that the mechanism of TMAO counter denaturation is simply that water and urea prefer to solvate TMAO rather than the protein, hence inhibiting its unfolding.     

The influence of urea and trimethylamine-N-oxide on hydrophobic interactions

Molecular dynamics simulations are used to obtain potentials of mean force for pairs of neopentane molecules immersed in aqueous solutions containing urea, trimethylamine-N-oxide (TMAO), or both solutes at once. It is shown that the hydrophobic attraction acting between neopentane pairs in pure water and in water-urea solution is completely destroyed by the addition of TMAO. This strongly suggests that TMAO does not counter the protein denaturing effect of urea by enhancing hydrophobic attraction amongst nonpolar groups.     

Osmolyte Effects on the Conformational Dynamics of a DNA Hairpin at Ambient and Extreme Environmental Conditions

The structural dynamics of a DNA hairpin (Hp) are studied in the absence and presence of the two natural osmolytes trimethylamine‐N‐oxide (TMAO) and urea at ambient and extreme environmental conditions, including high pressures and high temperatures, by using single‐molecule Förster resonance energy transfer and fluorescence correlation spectroscopy. The effect of pressure on the conformational dynamics of the DNA Hp is investigated on a single‐molecule level, providing novel mechanistic insights into its conformational conversions. Different from canonical DNA duplex structures of similar melting points, the DNA Hp is found to be rather pressure sensitive. The combined temperature and pressure dependent data allow dissection of the folding free energy into its enthalpic, entropic, and volumetric contributions. The folded conformation is effectively stabilized by the compatible osmolyte TMAO not only at high temperatures, but also at high pressures and in the presence of the destabilizing co‐solute urea.     

Modulation of the Thermodynamic Signatures of an RNA Thermometer by Osmolytes and Salts

Folding of ribonucleic acids (RNAs) is driven by several factors, such as base pairing and stacking, chain entropy, and ion‐mediated electrostatics, which have been studied in great detail. However, the power of background molecules in the cellular milieu is often neglected. Herein, we study the effect of common osmolytes on the folding equilibrium of a hairpin‐structured RNA and, using pressure perturbation, provide novel thermodynamic and volumetric insights into the modulation mechanism. The presence of TMAO causes an increased thermal stability and a more positive volume change for the helix‐to‐coil transition, whereas urea destabilizes the hairpin and leads to an increased expansibility of the unfolded state. Further, we find a strong interplay between water, salt, and osmolyte in driving the thermodynamics and defining the temperature and pressure stability limit of the RNA. Our results support a universal working mechanism of TMAO and urea to (de)stabilize proteins and the RNA.     

Thermodynamic characterization of the osmolyte effect on protein stability and the effect of GdnHCl on the protein denatured state

To understand the biomolecular interactions of osmolytes or guanidine hydrochloride (GdnHCl) with protein functional groups, we have determined the apparent transfer free energies (Delta'(tr)) of a homologous series of cyclic dipeptides (CDs) from water to aqueous solutions of osmolytes or GdnHCl through solubility measurements, as a function of osmolyte or GdnHCl concentration at 25 degrees C under atmospheric pressure. The materials investigated in the present study included the CDs of cyclo(Gly-Gly), cyclo(Ala-Gly), cyclo(Ala-Ala), cyclo(Leu-Ala), and cyclo(Val-Val), the osmolytes of trimethylamine N-oxide (TMAO), sarcosine, betaine, proline, and sucrose, and the denaturant of GdnHCl. We observed positive values of (Delta'(tr)) for CDs from water to osmolyte, indicating that interactions between osmolytes and CDs are unfavorable. In contrast, negative (Delta'(tr)) contributions were observed for CDs from water to GdnHCl, revealing that favorable interactions are predominant. The experimental results were further used to estimate the transfer free energies (Delta'(tr)) of the peptide bond (-CONH-), the peptide backbone unit (-CH2C=ONH-), and various functional groups from water to aqueous solutions of osmolyte or GdnHCl.     

Effect of urea on the aqueous solubility of dispersed dyes

Summary The solubilities of the following compounds in water and urea aqueous solutions were determined at 25‡ and 35 ‡C: diphenyl, azobenzene, p-aminoazobenzene, N,N-di(2-hydroxyethyl)-aminoazobenzene, N,N-dimethyl-p-aminobenzene and 1-phenylazo-4-aminonaphthalene. From the results the thermodynamic parameters for the transfer of one mole of the compound from water to urea aqueous solution were calculated. It was found that the process was nearly athermal; furthermore, it was invariably accompanied by a positive unitary entropy change. The mode of action of urea on the solubility of hydrophobic compounds in water was explained in terms of iceberg breaking ability of urea.

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Zusammenfassung Die L?slichkeiten folgender Verbindungen wurden in Wasser und w?\rigen Harnstoffl?sungen bei 25 und 35 ‡C bestimmt: N,N-di(2-Hydroxy?thyl)p-aminoazo-benzole, N,N-dimethyl-p-aminoazobenzole und 1-phenylazo-4-aminonaphthalm. Aus diesen Ergebnissen wurden die thermodynamischen Parameter für den übergang eines Mols der Verbindung aus Wasser in die Harnstoffl?sung berechnet. Der Proze\ verl?uft fast athermisch. Er ist au\erdem immer von einer positiven Entropie?nderung begleitet. Die Art der Wirkung des Harnstoffes auf die L?slichkeit hydrophober Verbindungen in Wasser wird auf Grund der F?higkeit des Harnstoffes, die Eisstruktur zu brechen, erkl?rt.

     

The molecular mechanism of stabilization of proteins by TMAO and its ability to counteract the effects of urea

Trimethylamine n-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins and offsets the destabilizing effects of urea. To investigate the molecular mechanism of these effects, we have studied the thermodynamics of interaction between TMAO and protein functional groups. The solubilities of a homologous series of cyclic dipeptides were measured by differential refractive index and the dissolution heats were determined calorimetrically as a function of TMAO concentration at 25 degrees C. The transfer free energy of the amide unit (-CONH-) from water to 1 M TMAO is large and positive, indicating an unfavorable interaction between the TMAO solution and the amide unit. This unfavorable interaction is enthalpic in origin. The interaction between TMAO and apolar groups is slightly favorable. The transfer free energy of apolar groups from water to TMAO consists of favorable enthalpic and unfavorable entropic contributions. This is in contrast to the contributions for the interaction between urea and apolar groups. Molecular dynamics simulations were performed to provide a structural framework for the interpretation of these results. The simulations show enhancement of water structure by TMAO in the form of a slight increase in the number of hydrogen bonds per water molecule, stronger water hydrogen bonds, and long-range spatial ordering of the solvent. These findings suggest that TMAO stabilizes proteins via enhancement of water structure, such that interactions with the amide unit are discouraged.     

Does Urea Alter the Collective Hydrogen‐Bond Dynamics in Water? A Dielectric Relaxation Study in the Terahertz‐Frequency Region

We report the ultrafast collective hydrogen‐bond dynamics of water in the extended hydration layer of urea by using terahertz time‐domain spectroscopy in the frequency region of 0.3–2.0 THz. The complex dielectric function has been fitted using a Debye relaxation model, and the timescales obtained are in the order of approximately 9 ps and 200 fs for bulk water; this exhibits a considerable acceleration beyond the 4 M urea concentration and indicates a possible disruption in the collective hydrogen‐bonded water‐network structure, which, in turn, provides an indirect support for the water “structure‐breaking” ability of urea. With 5 M urea in the presence of different concentrations of trimethylamine‐N‐oxide (TMAO), it was found that these parameters essentially follow the trend observed for TMAO itself, which signifies that any possible disruption of the water structure by urea is outdone by the strong hydrogen‐bonding ability of TMAO, which explains its ability to revive urea‐denatured proteins to their respective native states.     

尿素/氧化三甲胺混合溶剂影响单壁碳纳米管内部水合性质的分子动力学模拟

尿素是早已被人们认识的蛋白质变性剂,而氧化三甲胺则是最常用的蛋白质结构保护剂。虽然多年来被广泛应用在生物实验中,但是它们是如何在蛋白质结构形成中起作用,特别是氧化三甲胺是如何在高浓度尿素环境中起到抑制尿素蛋白变性作用的分子机制,至今仍然没有得到圆满解答。本文以单壁碳纳米管为模型疏水体系,采用分子动力学模拟研究尿素/氧化三甲胺混合溶液中纳米管内部水合性质,结果表明氧化三甲胺更易与水分子和尿素分子形成较强相互作用从而稳定了水溶液结构,这一结果亦表明了氧化三甲胺可以通过间接机制抵消尿素分子对于碳纳米管内部水合性质的影响。     

Electrodeposition of zinc coatings from the solutions of zinc oxide in imidazolium chloride/urea mixtures

To solve the inherent disadvantages in conventional processes for electrodeposition of zinc, it’s necessary to develop more high-efficiency and environmentally friendly electrolytes. In this work, it was found that the dissolution of ZnO was remarkably enhanced in some imidazolium chloride by the addition of urea, and the solubility of ZnO in 1:1 [Amim]Cl/urea mixture was as high as 8.35 wt% at 373.2 K. Electrochemical measurements showed that zinc could be readily electrodeposited from the solutions of ZnO. Bright, dense and well adherent zinc coatings with good purity were obtained from 0.6 M solution of ZnO in 1:1 [Amim]Cl/urea at 323.2 343.2 K. It’s expected that the solutions of ZnO in imidazolium chloride/urea mixtures have the potential to replace the traditional electrolytes, especially toxic zinc chloride-based ones for zinc electroplating, as well as preparation of zinc materials.     

Enthalpies of solution of urea in water-methanol mixtures at 298.15 K

The standard enthalpies of solution of urea in water-methanol mixtures were determined over the entire range of solvent compositions at 298.15 K. A comparison of the results obtained with the published data on mixtures of water with ethanol and n-propanol revealed a differentiating effect of the alcohol concentration on the enthalpy of solution of urea. Only for water-methanol solutions, an increase in the alkanol content in the mixture (0.6 mole fractions < x ₂ < 1.0 mole fractions) caused an increase in the degree of solvation of urea.