Hydrophobic Free Energy Research Articles

Surfactant Self-Assembly in Oppositely Charged Polymer Networks. Theory

The interaction of ionic surfactants with polyion networks of opposite charge in an aqueous environment is analyzed theoretically by applying a recent theory of surfactant ion-polyion complex salts (J. Colloid. Int. Sci. 2009, 332, 183). The theory takes into account attractive and repulsive polyion-mediated interactions between the micelles, the deformation of the polymer network, the mixing of micelles, polyion chains, and simple ions with water, and the hydrophobic free energy at the micelle surface. The theory is used to calculate binding isotherms, swelling isotherms, surfactant aggregation numbers, compositions of complexes,and phase structure under various conditions. Factors controlling the gel volume transition and conditions for core/shell phase coexistence are investigated in detail, as well as the influence of salt. In particular, the interplay between electrostatic and elastic interactions is highlighted. Results from theory are compared with experimental data reported in the literature. The agreement is found to be semiquantitative or qualitative. The theory explains both the discrete volume transition observed in systems where the surfactant is in excess over the polyion and the core/shell phase coexistence in systems where the polyion is in excess.

Relationship between β4 hydrogen bond and β6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S

Oversaturated deoxy-α 2β 2 T4V aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-α 2β 2 E6V,D73H. Solubility of deoxy-α 2β 2 T4V was ∼10-fold lower than that of deoxy-HbS and was similar to oxy- and deoxy-α 2β 2 E6V,T4V. These results indicate that β4Val in HbA in the oxy and deoxy forms with or without β6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a β4/β73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(α 2β 2 E6V,D73N), while α 2β 2 T4V and α 2β 2 D73H as well as HbS, α 2β 2 E6V,D73H and α 2β 2 E6V,T4V in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of β6 amino acid hydrophobicity that the synergism between the β4Thr hydrogen bond and β6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition.

The Human Cytomegalovirus UL44 C Clamp Wraps around DNA

Processivity factors tether the catalytic subunits of DNA polymerases to DNA so that continuous synthesis of long DNA strands is possible. The human cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer intermediate in structure between monomeric herpes simplex virus UL42, which binds DNA directly via a basic surface, and the trimeric sliding clamp PCNA, which encircles DNA. To investigate how UL44 interacts with DNA, calculations were performed in which a 12 bp DNA oligonucleotide was docked to UL44. The calculations suggested that UL44 encircles DNA, which interacts with basic residues both within the cavity of the C clamp and in flexible loops of UL44 that complete the "circle." The results of mutational and crosslinking studies were consistent with this model. Thus, UL44 is a "hybrid" of UL42 and PCNA: its structure is intermediate between the two and its mode of interaction with DNA has elements of both.

Modeling Sorption of Anionic Surfactants onto Sediment Materials: An a priori Approach for Perfluoroalkyl Surfactants and Linear Alkylbenzene Sulfonates

A mechanistically derived model predicting the sorption of anionic surfactants to sediments was developed and evaluated for three classes of surfactants: perfluoroalkyl carboxylates, perfluoroalkyl sulfonates, and linear alkylbenzene sulfonates. The model includes both hydrophobic and electrostatic components and estimates the contribution of each to the sediment-water distribution coefficient (Kd) using Gibbs free energy terms. The hydrophobic free energy term was calculated from the aqueous solubilities of non-charged alkylbenzene or perfluoroalkane analogs and prior observations of increases in Kd values with increasing chain lengths. The electrostatic term was calculated from aqueous solution measurements using the non-ideal competitive adsorption Donnan (NICA-Donnan) model. The NICA-Donnan calculations were performed using parameters previously derived for generic humic acids. These two terms were coupled by multiplying by the fraction of organic carbon in the sediment, foc, and a single fitting parameter, Faccess, the volumetric fraction of organic carbon accessible to the sorbing surfactant. The combined model accurately predicted the sediment-water distribution coefficients for all three classes of anionic surfactants. In its current formulation, the model was unable to capture the slight degree of isotherm nonlinearity observed for these surfactants.

Entropically-driven binding of mithramycin in the minor groove of C/G-rich DNA sequences

The antitumour antibiotic mithramycin A (MTA) is a DNA minor-groove binding ligand. It binds to C/G-rich tracts as a dimer that forms in the presence of divalent cations such as Mg2+. Differential scanning calorimetry, UV thermal denaturation, isothermal titration calorimetry and competition dialysis were used, together with computations of the hydrophobic free energy of binding, to determine the thermodynamic profile of MTA binding to DNA. The results were compared to those obtained in parallel using the structurally related mithramycin SK (MSK). The binding of MTA to salmon testes DNA determined by UV melting studies (Kobs = 1.2 (±0.3) × 105 M−1) is tighter than that of MSK (2.9 (±1.0) × 104 M−1) at 25°C. Competition dialysis studies showed a tighter MTA binding to both salmon testes DNA (42% C + G) and Micrococcus lysodeikticus DNA (72% C + G). The thermodynamic analysis of binding data at 25°C shows that the binding of MTA and MSK to DNA is entropically driven, dominated by the hydrophobic transfer of the antibiotics from solution to the DNA-binding site. Direct molecular recognition between MTA or MSK and DNA through hydrogen bonding and van der Waals contacts may also contribute significantly to complex formation.

Clustering of protein structures using hydrophobic free energy and solvent accessibility of proteins

The hydrophobic free energy and solvent accessibility of amino acids are used to study the relationship between the primary structure and structural classification of large proteins. A measure representation and a Z curve representation of protein sequences are proposed. Fractal analysis of the measure and Z curve representations of proteins and multifractal analysis of their hydrophobic free energy and solvent accessibility sequences indicate that the protein sequences possess correlations and multifractal scaling. The parameters from the fractal and multifractal analyses on these sequences are used to construct some parameter spaces. Each protein is represented by a point in these spaces. A method is proposed to distinguish and cluster proteins from the alpha, beta, alpha + beta, and alpha/beta structural classes in these parameter spaces. Fisher's linear discriminant algorithm is used to give a quantitative assessment of our clustering on the selected proteins. Numerical results indicate that the discriminant accuracies are satisfactory. In particular, they reach 94.12% and 88.89% in separating proteins from {alpha, alpha + beta, alpha/beta} proteins in a three-dimensional space.

Colloquium: Scaled particle theory and the length scales of hydrophobicity

Hydrophobic hydration plays a crucial role in self-assembly processes over multiple length scales, from the microscopic origins of inert gas solubility in water, to the mesoscopic organization of proteins and surfactant structures, to macroscopic phase separation. Many theoretical studies focus on the molecularly detailed interactions between oil and water, but the extrapolation of molecular-scale models to larger-length-scale hydration phenomena is sometimes not warranted. Scaled particle theories are based upon an interpolative view of that $\text{microscopic}\ensuremath{\leftrightarrow}\text{macroscopic}$ issue. This Colloquium revisits the scaled particle theory proposed 30 years ago by Stillinger [J. Solution Chem. 2, 141 (1973)], adopts a practical generalization, and considers the implications for hydrophobic hydration in light of our current understanding. The generalization is based upon identifying a molecular length, implicit in previous applications of scaled particle models, which provides an effective radius for joining microscopic and macroscopic descriptions. It will be demonstrated that the generalized theory correctly reproduces many of the anomalous thermodynamic properties of hydrophobic hydration for molecularly sized solutes, including solubility minima and entropy convergence, successfully interpolates between the microscopic and macroscopic extremes, and provides new insights into the underlying molecular mechanisms. The model considered here serves as a reference for theories that bridge microscopic and macroscopic hydrophobic effects. The results are discussed in terms of length scales associated with component phenomena. In particular, first there is a discussion of the microscopic-macroscopic joining radius identified by the theory; then follows a discussion of the Tolman length that describes curvature corrections to a surface area model of hydrophobic hydration free energies and the length scales on which entropy convergence of hydration free energies are expected.

Quantification of the adhesion free energy between bacteria and hydrophobic and hydrophilic substrata

In this paper, the bacterial adhesion to hydrophilic glass and hydrophobic indium tin oxide (ITO) coated glass surfaces was investigated. The aim of this work was to examine the effect of the hydrophobicity of two bacterial strains ( Pseudomonas stutzeri PS, and Staphylococcus epidermidis SE) and substrata surfaces on the adhesion mechanism. The hydrophobicity of the surfaces of the bacterial strains as well as of the surfaces of the tested materials was characterized using the contact angle measurement via sessile drop technique. The water contact angle measurements of the intrinsic cell hydrophobicity showed that the PS strain is more hydrophobic ( θ = 40°) than SE strain ( θ = 21°). For both tested materials, this angle was 12° for glass surface and 84° for ITO-coated glass surface. Based on the thermodynamic approach, the Lifshitz van der Waals interaction free energy, the hydrophobic interaction free energy and the total interaction free energy between the bacteria and the substratum through the suspending medium were quantified. In order to verify the thermodynamic approach predictions of the bacterial adhesion on each substratum, adhesion tests were carried out for each bacteria/substratum combination. The results revealed that for both materials surfaces, the adhesion energy of the hydrophobic PS strain (26.1 mJ/m 2 for glass and − 3.8 mJ/m 2 for ITO-coated glass) is higher than that of SE strain (31.8 mJ/m 2 for glass and 14.3 mJ/m 2 for ITO-coated glass). For both bacterial strains, the effect of the hydrophobicity property of the substratum seems more important in the initial adhesion step. However, the second step of adhesion involves the biological approach, since a discrepancy was found between physicochemical theoretical approach and adhesion tests for SE strain.

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 A) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.

Molecular modeling of mono- and bis-quaternary ammonium salts as ligands at the α4β2 nicotinic acetylcholine receptor subtype using nonlinear techniques

The neuronal nicotinic acetylcholine receptor (nAChR) has been a target for drug development studies for over a decade. A series of mono- and bis-quaternary ammonium salts, known to be antagonists at nAChRs, were separated into 3 structural classes and evaluated using both self-organizing map (SOM) and genetic functional approximation (GFA) algorithm models. Descriptors from these compounds were used to create several nonlinear quantitative structure-activity relationships (QSARs). The SOM methodology was effective in appropriately grouping these compounds with diverse structures and activities. The GFA models were also able to predict the activities of these molecules. Charge distribution and the hydrophobic free energies were found to be important indicators of bioactivity for this particular class of molecules. These QSAR approaches may be a useful to screen and select in silico new drug candidates from larger compound libraries to be further evaluated in in vitro biological assays.

Importance of main-chain hydrophobic free energy to the stability of thermophilic proteins

Living organisms are found in the most unexpected places, including deep-sea vents at 100 °C and several hundred bars pressure, in hot springs. Needless to say, the proteins found in thermophilic species are much more stable than their mesophilic counterparts. There are no obvious reasons to say that one would be more stable than others. Even examination of the amino acids and comparison of structural features of thermophiles with mesophilies cannot bring satisfactory explanation for the thermal stability of such proteins. In order to bring out the hidden information behind the thermal stabilization of such proteins in terms of energy factors and their combinations, analysis were made on good resolution structures of thermophilic and their mesophilic homologous from 23 different families. From the structural coordinates, free energy contributions due to hydrophobic, electrostatic, hydrogen bonding, disulfide bonding and van der Waals interactions are computed. In this analysis, a vast majority of thermophilic proteins adopt slightly lower free energy contribution in each energy terms than its mesophilic counterparts. The major observation noted from this study is the lower hydrophobic free energy contribution due to carbon atoms and main-chain nitrogen atoms in all the thermophilic proteins. The possible combination of different free energy terms shows majority of the thermophilic proteins have lower free energy strategy than their mesophilic homologous. The derived results show that the hydrophobic free energy due to carbon and nitrogen atoms and such combinations of free energy components play a vital role in the thermostablisation of such proteins.

Correlation of Three-dimensional Structures with the Antibacterial Activity of a Group of Peptides Designed Based on a Nontoxic Bacterial Membrane Anchor

To understand the functional differences between a nontoxic membrane anchor corresponding to the N-terminal sequence of the Escherichia coli enzyme IIA(Glc) and a toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was designed to bridge the gap between them. An alteration of a single residue of the membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra indicate that all peptides are disordered in water but helical in micelles. Structures of the peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy. The quality of the distance-based structures was improved by including backbone angle restraints derived from a set of chemical shifts ((1)H(alpha), (15)N, (13)C(alpha), and (13)C(beta)) from natural abundance two-dimensional heteronuclear correlated spectroscopy. Different from the membrane anchor, antibacterial peptides possess a broader and longer hydrophobic surface, allowing a deeper penetration into the membrane, as supported by intermolecular nuclear Overhauser effect cross-peaks between the peptide and short chain dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures of these peptides with their antibacterial activity. The activity of this group of peptides does not correlate exactly with helicity, amphipathicity, charge, the number of charges, the size of the hydrophobic surface, or hydrophobic transfer free energy. However, a correlation is established between the peptide activity and membrane perturbation potential, which is defined by interfacial hydrophobic patches and basic residues in the case of cationic peptides. Indeed, (31)P solid state NMR spectroscopy of lipid bilayers showed that the extent of lipid vesicle disruption by these peptides is proportional to their membrane perturbation potential.

Relative importance of secondary structure and solvent accessibility to the stability of protein mutants.: A case study with amino acid properties and energetics on T4 and human lysozymes

Understanding the factors influencing the stability of protein mutants is an important task in molecular and computational biology. In this work, we have approached this problem by examining the relative importance of secondary structure and solvent accessibility of the mutant residue for understanding/predicting the stability of protein mutants. We have used hydrophobic, electrostatic and hydrogen bond free energy terms and nine unique physicochemical, energetic and conformational properties of amino acids in the present study and these parameters have been related with changes in thermal stability (Δ T m) of all the single mutants of lysozymes based on single and multiple correlation coefficients. As expected the properties reflecting hydrophobicity and hydrophobic free energy play a major role to distinguish stabilizing and destabilizing mutants. The hydrophobic free energy due to carbon and nitrogen atoms distinguish the stability of coil and strand mutations to the accuracy of 100 and 90%, respectively. In agreement with previous results, the subgroup classification based on secondary structure and the information about its location in the structure yielded good relationship with the experimental Δ T m. We revealed that the secondary structure information is equally or more important than solvent accessibility for understanding the stability of protein mutants. The comparison of amino acid properties with free-energy terms indicate that the energetic contribution explains the mutant stability better in coil region whereas the amino acid properties do better in strand region. Further, the combination of free energies with amino acid properties increased the correlation significantly. The present study demonstrates the importance of classifying the mutants based on secondary structure to the stability of proteins upon mutations.

Role of non-covalent interactions for determining the folding rate of two-state proteins

Understanding the factors influencing the folding rate of proteins is a challenging problem. In this work, we have analyzed the role of non-covalent interactions for the folding rate of two-state proteins by free-energy approach. We have computed the free-energy terms, hydrophobic, electrostatic, hydrogen-bonding and van der Waals free energies. The hydrophobic free energy has been divided into the contributions from different atoms, carbon, neutral nitrogen and oxygen, charged nitrogen and oxygen, and sulfur. All the free-energy terms have been related with the folding rates of 28 two-state proteins with single and multiple correlation coefficients. We found that the hydrophobic free energy due to carbon atoms and hydrogen-bonding free energy play important roles to determine the folding rate in combination with other free energies. The normalized energies with total number of residues showed better results than the total energy of the protein. The comparison of amino acid properties with free-energy terms indicates that the energetic terms explain better the folding rate than amino acid properties. Further, the combination of free energies with topological parameters yielded the correlation of 0.91. The present study demonstrates the importance of topology for determining the folding rate of two-state proteins.

Testing the Gouy–Chapman theory by means of surface tension measurements for SDS–NaCl–H 2O mixtures

Surface tension isotherms were measured for sodium dodecyl sulfate (SDS) at different concentrations of added salt (NaCl). The free energy of the surfactant monolayer was assessed by invoking the Gouy–Chapman theory for the charged head groups, the hydrophobic (Tanford) free energy of transfer of the hydrocarbon chain, and the hydrocarbon chain configurational free energy according to Gruen's calculations and finally macroscopic contact terms. In particular, the effect of an increased salt concentration in bulk was examined. Theoretical predictions compare well with the experimental findings, and good agreement was found with respect to both the variation of free energy of the monolayer and the surface pressure behavior. Thus, at least for a liquid-expanded monolayer of SDS, the Gouy–Chapman model yields a satisfactory account of the electrostatic contribution to the thermodynamic properties at different salt concentrations of NaCl.

Long-range and short-range mechanisms of hydrophobic attraction and hydrophilic repulsion in specific and aspecific interactions.

Among the three different non-covalent forces acting in aqueous media, i.e. Lifshitz-van der Waals (LW), Lewis acid-base (AB) and electrical double layer (EL) forces, the AB forces or electron-acceptor/electron-donor interactions are quantitatively by far the predominant ones. A subset of the AB forces acting in water causes the hydrophobic effect, which is the attraction caused by the hydrogen-bonding (AB) free energy of cohesion between the water molecules which surround all apolar as well as polar molecules and particles when they are immersed in water. As the polar energy of cohesion among water molecules is an innate property of water, the hydrophobic attraction (due to the hydrophobic effect) is unavoidably always present in aqueous media and has a value of DeltaG(hydrophobic) = -102 mJ/m(2), at 20 degrees C, being equal to the AB free energy of cohesion between the water molecules at that temperature. The strong underlying hydrophobic attraction due to this effect can, however, be surmounted by very hydrophilic molecules and particles that attract water molecules more strongly than the free energy of attraction of these molecules or particles for one another, plus the hydrogen-bonding free energy of cohesion between the water molecules, thus resulting in a net non-electrical double layer repulsion. Each of the three non-covalent forces, LW, AB or EL, any of which can be independently attractive or repulsive, decays, dependent on the circumstances, as a function of distance according to different rules. These rules, following an extended DLVO (XDLVO) approach, are given, as well as the measurement methods for the LW, AB and EL surface thermodynamic properties, determined at "contact". The implications of the resulting hydrophobic attractive and hydrophilic repulsive free energies, as a function of distance, are discussed with respect to specific and aspecific interactions in biological systems. The discussion furnishes a description of the manner by which shorter-range specific attractions can surmount the usually much stronger long-range aspecific repulsion, and ends with examples of in vitro and in vivo effects of hydrophilization of biopolymers, particles or surfaces by linkage with polyethylene oxide (PEO; also called polyethylene glycol, PEG).

Entry in Emulsion Polymerization: Effects of Initiator and Particle Surface Charge

The rate coefficient for radical entry into latex particles in emulsion polymerizations is measured for styrene systems in which the entering species are anionic (from persulfate) and cationic (from 2,2‘-azobis(2-methylpropionamidine) dihydrochloride, or V-50). These entry rate coefficients ρ are obtained by measuring rates in seeded emulsion polymerizations where the seeds have either cationic or anionic groups on the surface; “zero-one” conditions are employed, because these offer the advantage that particle size is sufficiently small (≈70 nm diameter) that intraparticle termination is not rate-determining. Data comprise steady-state rates with chemical initiator, combined with loss rates obtained using γ-radiolysis initiation and following the relaxation behavior after removal from the radiation source. Values for ρ as a function of initiator concentration can be meaningfully compared for different initiators through the dependence of initiator efficiency fentry on primary radical generation rate (radical flux). For the anionic latex, this dependence is seen to differ depending on the nature of the initiator used. This may be explained by the entry model [Maxwell, I. A.; Morrison, B. R.; Napper, D. H.; Gilbert, R. G. Macromolecules 1991, 24, 1629] wherein the rate-determining steps in entry are assumed to be only aqueous-phase propagation and termination to form surface-active z-meric oligomeric radicals; entry is solely by z-mers, for which actual entry into the particle is so fast as not to be rate-determining. The cationic latex shows a high rate of spontaneous initiation, which can be explained in terms of amidino radical chemistry; this can be reduced by heat treatment. Accurate fentry values are obtained using the heat-treated cationically stabilized latex for seeded studies. The fentry data are also consistent with the model for both cationic and anionic species entering the cationic latex. Values of z so inferred (≈2 for persulfate, ≈1 for amidinium) can be understood in terms of the hydrophobic free energy of these species. The results refute alternative models in the literature that suppose entry is controlled by double-layer (colloidal) interactions, surfactant displacement, or diffusion control.

Protein Chemistry at Membrane Interfaces: Non-additivity of Electrostatic and Hydrophobic Interactions

Non-specific binding of proteins and peptides to charged membrane interfaces depends upon the combined contributions of hydrophobic (Δ G HΦ) and electrostatic (Δ G ES) free energies. If these are simply additive, then the observed free energy of binding (Δ G obs) will be given by Δ G obs=Δ G HΦ+Δ G ES, where Δ G HΦ=−σ NP A NP and Δ G ES= zFφ. In these expressions, A NP is the non-polar accessible area, σ NP the non-polar solvation parameter, z the formal peptide valence, F the Faraday constant, and φ the membrane surface potential. But several lines of evidence suggest that hydrophobic and electrostatic binding free energies of proteins at membrane interfaces, such as those associated with cell signaling, are not simply additive. In order to explore this issue systematically, we have determined the interfacial partitioning free energies of variants of indolicidin, a cationic proline-rich antimicrobial peptide. The synthesized variants of the 13 residue peptide covered a wide range of hydrophobic free energies, which allowed us to examine the effect of hydrophobicity on electrostatic binding to membranes formed from mixtures of neutral and anionic lipids. Although Δ G obs was always a linear function of Δ G HΦ, the slope depended upon anionic lipid content: the slope was 1.0 for pure, zwitterionic phosphocholine bilayers and 0.3 for pure phosphoglycerol membranes. Δ G obs also varied linearly with surface potential, but the slope was smaller than the expected value, zF. As observed by others, this suggests an effective peptide valence z eff that is smaller than the formal valence z. Because of our systematic approach, we were able to establish a useful rule-of-thumb: z eff is reduced relative to z by about 20 % for each 3 kcal mol −1 (1 kcal=4.184 kJ) favorable increase in Δ G HΦ. For neutral phosphocholine interfaces, we found that Δ G obs could be predicted with remarkable accuracy using the Wimley-White experiment-based interfacial hydrophobicity scale.

Scaling of Hydrophobic Solvation Free Energies

We have calculated the free energy of solvation for hard sphere solutes, as large as 20 A in diameter, in two simple-point-charge models of water. These results were obtained using umbrella sampling of ensembles with fixed, ambient temperature and pressure. For the same water models, we have also calculated the surface tension of a liquid−vapor interface at room temperature. Analogous calculations were carried out for three thermodynamic states of the Lennard-Jones (LJ) fluid near liquid−vapor coexistence. For both water and the LJ fluid at the conditions we have simulated, extrapolation of our results suggests that the planar interface between coexisting liquid and vapor phases has the same surface tension as the planar limit of hard sphere solvation. We expect this correspondence to be a general result for fluids at thermodynamic states close to phase coexistence, as measured by the difference in chemical potential between bulk liquid and vapor phases, and far from the critical point. The solvation free...

Molecular dynamics study on hydrophobic effects in aqueous urea solutions.

Hydrophobic effects in aqueous urea were analyzed by molecular dynamics simulations. The contribution of solvents to the potential of mean force between two methane molecules was calculated by using molecular dynamics simulations and was compared with the solubility data of hydrocarbons in aqueous urea. Both the simulation results and the solubility data indicated that urea stabilizes methane-methane association. The stabilization was due to increasing the solvation free energies of small hydrocarbons such as methane by addition of urea. The solvation free energies of larger hydrocarbons, on the other hand, are decreased by addition of urea. This effect of the solute size on hydrophobic free energies in aqueous urea was also analyzed by using molecular dynamics simulations by means of division of the solvation process into two parts: the cavity formation and the introduction of the solute-solvent attractive interactions. In the cavity formation, urea increased hydrophobic free energies, and in the introduction of the solute-solvent attractive interactions, urea decreased hydrophobic free energies. The influence of urea on hydrophobic free energies was determined by the balance of effects of the two parts of the solvation process.