Nanocrystalline protein domains via salting-out.

Protein salting-out is a well established phenomenon that in many cases leads to amorphous structures and protein gels, which are usually not considered to be useful for protein structure determination. Here, microstructural measurements of several different salted-out protein dense phases are reported, including of lysozyme, ribonuclease A and an IgG1, showing that salted-out protein gels unexpectedly contain highly ordered protein nanostructures that assemble hierarchically to create the gel. The nanocrystalline domains are approximately 10-100 nm in size, are shown to have structures commensurate with those of bulk crystals and grow on time scales in the order of an hour to a day. Beyond revealing the rich, hierarchical nanoscale to mesoscale structure of protein gels, the nanocrystals that these phases contain are candidates for structural biology on next-generation X-ray free-electron lasers, which may enable the study of biological macromolecules that are difficult or impossible to crystallize in bulk.

Autobiography of Stanley I. Sandler.

I review my career from its academic beginning to my recent retirement. I grew up and studied chemical engineering in New York City. My initial failure to understand thermodynamics the way it had been taught, evidenced by the difficulty I had when starting graduate school, led me years later to write a textbook on the subject that is now in a fifth edition, in addition to other books I have written. My research areas have included molecular simulation, statistical- and quantum mechanical-based methods, and a variety of experimental thermodynamic measurements. In addition, I have been a consultant in traditional chemical engineering areas, as well in nontraditional areas, such as assisting in the design of a heat shield for interplanetary exploration, the destruction of armed chemical weapons, and the cleanup of nuclear weapons production facilities.

Quantum Chemically Estimated Abraham Solute Parameters Using Multiple Solvent-Water Partition Coefficients and Molecular Polarizability.

Polyparameter Linear Free Energy Relationships (pp-LFERs), also called Linear Solvation Energy Relationships (LSERs), are used to predict many environmentally significant properties of chemicals. A method is presented for computing the necessary chemical parameters, the Abraham parameters (AP), used by many pp-LFERs. It employs quantum chemical calculations and uses only the chemical's molecular structure. The method computes the Abraham E parameter using density functional theory computed molecular polarizability and the Clausius-Mossotti equation relating the index refraction to the molecular polarizability, estimates the Abraham V as the COSMO calculated molecular volume, and computes the remaining AP S, A, and B jointly with a multiple linear regression using sixty-five solvent-water partition coefficients computed using the quantum mechanical COSMO-SAC solvation model. These solute parameters, referred to as Quantum Chemically estimated Abraham Parameters (QCAP), are further adjusted by fitting to experimentally based APs using QCAP parameters as the independent variables so that they are compatible with existing Abraham pp-LFERs. QCAP and adjusted QCAP for 1827 neutral chemicals are included. For 24 solvent-water systems including octanol-water, predicted log solvent-water partition coefficients using adjusted QCAP have the smallest root-mean-square errors (RMSEs, 0.314-0.602) compared to predictions made using APs estimated using the molecular fragment based method ABSOLV (0.45-0.716). For munition and munition-like compounds, adjusted QCAP has much lower RMSE (0.860) than does ABSOLV (4.45) which essentially fails for these compounds.

Predictions of the physicochemical properties of amino acid side chain analogs using molecular simulation.

A candidate drug compound is released for clinical trails (in vivo activity) only if its physicochemical properties meet desirable bioavailability and partitioning criteria. Amino acid side chain analogs play vital role in the functionalities of protein and peptides and as such are important in drug discovery. We demonstrate here that the predictions of solvation free energies in water, in 1-octanol, and self-solvation free energies computed using force field-based expanded ensemble molecular dynamics simulation provide good accuracy compared to existing empirical and semi-empirical methods. These solvation free energies are then, as shown here, used for the prediction of a wide range of physicochemical properties important in the assessment of bioavailability and partitioning of compounds. In particular, we consider here the vapor pressure, the solubility in both water and 1-octanol, and the air-water, air-octanol, and octanol-water partition coefficients of amino acid side chain analogs computed from the solvation free energies. The calculated solvation free energies using different force fields are compared against each other and with available experimental data. The protocol here can also be used for a newly designed drug and other molecules where force field parameters and charges are obtained from density functional theory.

Local Crystalline Structure in an Amorphous Protein Dense Phase.

Proteins exhibit a variety of dense phases ranging from gels, aggregates, and precipitates to crystalline phases and dense liquids. Although the structure of the crystalline phase is known in atomistic detail, little attention has been paid to noncrystalline protein dense phases, and in many cases the structures of these phases are assumed to be fully amorphous. In this work, we used small-angle neutron scattering, electron microscopy, and electron tomography to measure the structure of ovalbumin precipitate particles salted out with ammonium sulfate. We found that the ovalbumin phase-separates into core-shell particles with a core radius of ∼2 µm and shell thickness of ∼0.5 µm. Within this shell region, nanostructures comprised of crystallites of ovalbumin self-assemble into a well-defined bicontinuous network with branches ∼12 nm thick. These results demonstrate that the protein gel is comprised in part of nanocrystalline protein.

A comparative study of monoclonal antibodies. 1. Phase behavior and protein-protein interactions.

Protein phase behavior is involved in numerous aspects of downstream processing, either by design as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. This work explores the phase behavior of eight monoclonal antibodies (mAbs) that exhibit liquid-liquid separation, aggregation, gelation, and crystallization. The phase behavior has been studied systematically as a function of a number of factors, including solution composition and pH, in order to explore the degree of variability among different antibodies. Comparisons of the locations of phase boundaries show consistent trends as a function of solution composition; however, changing the solution pH has different effects on each of the antibodies studied. Furthermore, the types of dense phases formed varied among the antibodies. Protein-protein interactions, as reflected by values of the osmotic second virial coefficient, are used to correlate the phase behavior. The primary findings are that values of the osmotic second virial coefficient are useful for correlating phase boundary locations, though there is appreciable variability among the antibodies in the apparent strengths of the intrinsic protein-protein attraction manifested. However, the osmotic second virial coefficient does not provide a clear basis to predict the type of dense phase likely to result under a given set of solution conditions.

Monte Carlo simulations for the free energies of C60 and C70 fullerene crystals by acceptance ratio method and expanded ensemble method.

Accurate values of the free energies of C60 and C70 fullerene crystals are obtained using expanded ensemble method and acceptance ratio method combined with the Einstein-molecule approach. Both simulation methods, when tested for Lennard-Jones crystals, give accurate results of the free energy differing from each other in the fifth significant digit. The solid-solid phase transition temperature of C60 crystal is determined from free energy profiles, and found to be 260 K, which is in good agreement with experiment. For C70 crystal, using the potential model of Sprik et al. [Phys. Rev. Lett. 69, 1660 (1992)], low-temperature solid-solid phase transition temperature is found to be 160 K determined from the free energy profiles. Whereas this is somewhat lower than the experimental value, it is in agreement with conventional molecular simulations, which validates the methodological consistency of the present simulation method. From the calculations of the free energies of C60 and C70 crystals, we note the significance of symmetry number for crystal phase needed to properly account for the indistinguishability of orientationally disordered states.

Anisotropic Contributions to Protein-Protein Interactions.

The anisotropy of shape and functionality of proteins complicates the prediction of protein-protein interactions. We examine the distribution of electrostatic and nonelectrostatic contributions to these interactions for two globular proteins, lysozyme and chymosin B, which differ in molecular weight by about a factor of 2. The interaction trends for these proteins are computed in terms of contributions to the osmotic second virial coefficient that are evaluated using atomistic models of the proteins. Our emphasis is on identifying the orientational configurations that contribute most strongly to the overall interactions due to high-complementarity interactions, and on calculating the effect of ionic strength on such interactions. The results emphasize the quantitative importance of several features of protein interactions, notably that despite differences in their frequency of occurrence, configurations differing appreciably in interaction energy can contribute meaningfully to overall interactions. However, relatively small effects due to charge anisotropy or specific hydration can affect the overall interaction significantly only if they contribute to strongly attractive configurations. The results emphasize the necessity of accounting for detailed anisotropy to capture actual experimental trends, and the sensitivity of even very detailed atomistic models to subtle solution contributions.

Adsorption of HMF from water/DMSO solutions onto hydrophobic zeolites: experiment and simulation.

The adsorption of 5-hydroxymethylfurfural (HMF), DMSO, and water from binary and ternary mixtures in hydrophobic silicalite-1 and dealuminated Y (DAY) zeolites at ambient conditions was studied by experiments and molecular modeling. HMF and DMSO adsorption isotherms were measured and compared to those calculated using a combination of grand canonical Monte Carlo and expanded ensemble (GCMC-EE) simulations. A method based on GCMC-EE simulations for dilute solutions combined with the Redlich-Kister (RK) expansion (GCMC-EE-RK) is introduced to calculate the isotherms over a wide range of concentrations. The simulations, using literature force fields, are in reasonable agreement with experimental data. In HMF/water binary mixtures, large-pore hydrophobic zeolites are much more effective for HMF adsorption but less selective because large pores allow water adsorption because of H2 O-HMF attraction. In ternary HMF/DMSO/water mixtures, HMF loading decreases with increasing DMSO fraction, rendering the separation of HMF from water/DMSO mixtures by adsorption difficult. The ratio of the energetic interaction in the zeolite to the solvation free energy is a key factor in controlling separation from liquid mixtures. Overall, our findings could have an impact on the separation and catalytic conversion of HMF and the rational design of nanoporous adsorbents for liquid-phase separations in biomass processing.

Comparison of homogeneous and heterogeneous catalysts for glucose-to-fructose isomerization in aqueous media.

Herein, the first comparison of the mechanisms of glucose-to-fructose isomerization in aqueous media enabled by homogeneous (CrCl3 and AlCl3 ) and heterogeneous catalysts (Sn-beta) by using isotopic-labeling studies is reported. A pronounced kinetic isotope effect (KIE) was observed if the deuterium label was at the C2 position, thus suggesting that a hydrogen shift from the C2 to C1 positions was the rate-limiting step with the three catalysts. (13) C and (1) H NMR spectroscopic investigations confirmed that an intra-hydride-transfer reaction pathway was the predominant reaction channel for all three catalysts in aqueous media. Furthermore, the deuterium atom in the labeled glucose could be mapped onto hydroxymethylfurfural and formic acid through reactions that followed the isomerization step in the presence of Brønsted acids. In all three catalysts, the active site appeared to be a bifunctional Lewis-acidic/Brønsted-basic site, based on a speciation model and first-principles calculations. For the first time, a mechanistic similarities between the homogeneous and heterogeneous catalysis of aldose-to-ketose isomerization is established and it is suggested that learning from homogeneous catalysis could assist in the development of improved heterogeneous catalysts.

Isosteric heats of gas and liquid adsorption.

The heat of adsorption is an indicator of the strength of the interaction between an adsorbate and a solid adsorbent. This parameter can be determined from the heat released in calorimetric experiments or from the analysis of adsorption isotherms at different temperatures. The latter, called isosteric heats of adsorption, are commonly used in the characterization of materials for gas- and liquid-phase adsorption. Although the equations for the determination of isosteric heats of adsorption from the gas phase are well-known, approximate equations are frequently used for liquid-phase adsorption. We present here the rigorous equations for determining the isosteric heats of gas- and liquid-phase adsorption and their relation to the commonly used approximate equations. These equations are used to compute the isosteric heats of liquid adsorption based on the adsorption isotherms obtained from simulations for two well-defined systems, one ideal and the other nonideal. The results of using the rigorous equations are compared with those from the approximate equations. The main conclusion is that the commonly used approximate equations provide reasonable, but not perfect, estimates of the isosteric heats of liquid adsorption using only the experimental adsorption isotherms. The more accurate rigorous equations require additional information, including the heat of vaporization and, for nonideal mixtures, the heat of mixing.

Measurement of activity coefficients of mixtures by head-space gas chromatography: general procedure.

Head-space gas chromatography (HS-GC) is an applicable method to perform vapor-liquid equilibrium measurements and determine activity coefficients. However, the reproducibility of the data may be conditioned by the experimental procedure concerning to the automated pressure-balanced system. The study developed in this work shows that a minimum volume of liquid in the vial is necessary to ensure the reliability of the activity coefficients since it may become a parameter that influences the magnitude of the peak areas: the helium introduced during the pressurization step may produce significant variations of the results when too small volume of liquid is selected. The minimum volume required should thus be evaluated prior to obtain experimentally the concentration in the vapor phase and the activity coefficients. In this work, the mixture acetonitrile-toluene is taken as example, requiring a sample volume of more than 5mL (about more than 25% of the vial volume). The vapor-liquid equilibrium and activity coefficients of mixtures at different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 molar fraction) and four temperatures (35, 45, 55 and 70°C) have been determined. Relative standard deviations (RSD) lower than 5% have been obtained, indicating the good reproducibility of the method when a sample volume larger than 5mL is used. Finally, a general procedure to measure activity coefficients by means of pressure-balanced head-space gas chromatography is proposed.

Insights into the interplay of Lewis and Brønsted acid catalysts in glucose and fructose conversion to 5-(hydroxymethyl)furfural and levulinic acid in aqueous media.

5-(Hydroxymethyl)furfural (HMF) and levulinic acid production from glucose in a cascade of reactions using a Lewis acid (CrCl3) catalyst together with a Brønsted acid (HCl) catalyst in aqueous media is investigated. It is shown that CrCl3 is an active Lewis acid catalyst in glucose isomerization to fructose, and the combined Lewis and Brønsted acid catalysts perform the isomerization and dehydration/rehydration reactions. A CrCl3 speciation model in conjunction with kinetics results indicates that the hydrolyzed Cr(III) complex [Cr(H2O)5OH](2+) is the most active Cr species in glucose isomerization and probably acts as a Lewis acid-Brønsted base bifunctional site. Extended X-ray absorption fine structure spectroscopy and Car-Parrinello molecular dynamics simulations indicate a strong interaction between the Cr cation and the glucose molecule whereby some water molecules are displaced from the first coordination sphere of Cr by the glucose to enable ring-opening and isomerization of glucose. Additionally, complex interactions between the two catalysts are revealed: Brønsted acidity retards aldose-to-ketose isomerization by decreasing the equilibrium concentration of [Cr(H2O)5OH](2+). In contrast, Lewis acidity increases the overall rate of consumption of fructose and HMF compared to Brønsted acid catalysis by promoting side reactions. Even in the absence of HCl, hydrolysis of Cr(III) decreases the solution pH, and this intrinsic Brønsted acidity drives the dehydration and rehydration reactions. Yields of 46% levulinic acid in a single phase and 59% HMF in a biphasic system have been achieved at moderate temperatures by combining CrCl3 and HCl.

Comparison of two simulation methods to compute solvation free energies and partition coefficients.

The thermodynamic integration (TI) and expanded ensemble (EE) methods are used here to calculate the hydration free energy in water, the solvation free energy in 1-octanol, and the octanol-water partition coefficient for a six compounds of varying functionality using the optimized potentials for liquid simulations (OPLS) all-atom (AA) force field parameters and atomic charges. Both methods use the molecular dynamics algorithm as a primary component of the simulation protocol, and both have found wide applications in fields such as the calculation of activity coefficients, phase behavior, and partition coefficients. Both methods result in solvation free energies and 1-octanol/water partition coefficients with average absolute deviations (AAD) from experimental data to within 4 kJ/mol and 0.5 log units, respectively. Here, we find that in simulations the OPLS-AA force field parameters (with fixed charges) can reproduce solvation free energies of solutes in 1-octanol with AAD of about half that for the solute hydration free energies using a extended simple point charge (SPC/E) model of water. The computational efficiency of the two simulation methods are compared based on the time (in nanoseconds) required to obtain similar standard deviations in the solvation free energies and 1-octanol/water partition coefficients. By this analysis, the EE method is found to be a factor of nine more efficient than the TI algorithm. For both methods, solvation free energy calculations in 1-octanol consume roughly an order of magnitude more CPU hours than the hydration free energy calculations.

Physicochemical Properties of Hazardous Energetic Compounds from Molecular Simulation.

A protocol is presented and used for the computation of physicochemical properties of nitroaromatic energetic compounds (ECs) using molecular simulation. Solvation and self-solvation free energies of ECs are computed using an expanded ensemble (EE) molecular dynamics method, with the TraPPE-UA/CHELPG and CGenFF/CHELPG force field models. Thermodynamic pathways relating Gibbs free energies and physicochemical properties are used to predict the room temperature vapor pressures, solubilities (in water and 1-octanol), Henry's law constants, and partition coefficients (octanol-water, air-water, and air-octanol) for liquid, subcooled, and solid ECs from the molecular simulations. These predictions are compared to experimental data where available. It is found that the use of the TraPPE-UA model with CHELPG charges computed here leads to predictions of measured physicochemical properties of comparable accuracy to that of other theoretical and empirical models. However, the advantage of the method used here is that with no experimental data, unlike other methods, a number of physicochemical properties for a compound can be calculated from only its atomic connectivity, charges obtained from density function theory (DFT), and choice of force field using two simulations: its self-solvation free energy and its Gibbs free energy in a solvent.

Hydration Free Energies of Multifunctional Nitroaromatic Compounds.

Nitroaromatic compounds (NACs) are used as energetic materials, reagents, and pesticides; however, they are potentially hazardous for the environment and human health. To predict the environmental distribution of these compounds, the vapor pressure, aqueous solubility, and Henry's law constant are important properties, as is the solvation free energy in water from which the latter two can be computed. Here, we have calculated the hydration free energies for a set of nine nitroaromatic compounds containing one, two, and three nitro groups using the expanded ensemble molecular dynamics simulation method with TIP3P water and the GAFF, CGenFF, OPLS-AA, and TraPPE force field parameters and the RESP (gas phase), CHELPG (gas phase), and CM4 (aqueous phase) partial atomic charges calculated here. Also, we have computed hydration free energies using the reported default partial atomic charges of the OPLS-AA force field and using the semiempirical AM1-BCC charges with GAFF parameters. The effect of water model flexibility on the computation of hydration free energy is examined with CGenFF/(CHELPG+SPC-Fw) model. All the force fields studied generally led to less accurate predictions with increasing numbers of nitro groups. The average unsigned errors (AUE) show that 6 of 16 force-field/(charge+water) models used perform approximately equally well in predicting measured hydration free energies: these are CGenFF/(CHELPG+TIP3P), CGenFF/(CM4+TIP3P), OPLS-AA/(CHELPG+TIP3P), OPLS-AA/(CM4+TIP3P), TraPPE-UA/(CHELPG+TIP3P), and TraPPE-UA/(CM4+TIP3P). When using the default atomic charges, the OPLS-AA force field was the most accurate, though using CHELPG and CM4 charges led to better predictions. Our analyses indicate that not only the charges but also the van der Waals interaction parameters for the nitro-group nitrogen and oxygen atoms in the force fields are partly responsible for the performance variations in predicting solvation free energies. We also compared the force field-based simulation results with the predictions from the SM6 solvation model and Abraham linear solvation energy relationship (LSER) method. With an appropriate choice of theory and basis set both for geometry optimization and computation, which unfortunately is not known a priori, the SM6 model hydration free energy predictions for the NACs are comparable to the simulation results here. The Abraham LSER predictions with descriptors obtained from the Platts method are of reasonable accuracy. A useful addition to this paper is the Supporting Information that contains a compiled and evaluated list of the hydration free energies of the NACs studied here assembled from the literature.

Solvation free energies and hydration structure of N-methyl-p-nitroaniline.

Solvation Gibbs energies of N-methyl-p-nitroaniline (MNA) in water and 1-octanol are calculated using the expanded ensemble molecular dynamics method with a force field taken from the literature. The accuracy of the free energy calculations is verified with the experimental Gibbs free energy data and found to reproduce the experimental 1-octanol∕water partition coefficient to within ±0.1 in log unit. To investigate the hydration structure around N-methyl-p-nitroaniline, an independent NVT molecular dynamics simulation was performed at ambient conditions. The local organization of water molecules around the solute MNA molecule was investigated using the radial distribution function (RDF), the coordination number, and the extent of hydrogen bonding. The spatial distribution functions (SDFs) show that the water molecules are distributed above and below the nitrogen atoms parallel to the plane of aromatic ring for both the methylamino and nitro functional groups. It is found that these groups have a significant effect on the hydration of MNA with water molecules forming two weak hydrogen bonds with both the methylamino and nitro groups. The hydration structures around the functional groups in MNA in water are different from those that have been found for methylamine, nitrobenzene, and benzene in aqueous solutions, and these differences together with weak hydrogen bonds explain the lower solubility of MNA in water. The RDFs together with SDFs provide a tool for the understanding the hydration of MNA (and other molecules) and therefore their solubility.

Molecular screening of alcohol and polyol adsorption onto MFI-type zeolites.

Configurational-bias grand canonical Monte Carlo (CB-GCMC) simulations and expanded ensemble (EE)-CB-GCMC simulations were performed to obtain adsorption isotherms of alcohols and polyols onto MFI-type zeolites from the gas phase and aqueous solution. In adsorption from both phases, Henry's constants and heats of adsorption at infinite dilution for straight-chain alcohols, diols, and triols in silicalite-1 are found to increase, and the saturation loadings decrease with increasing carbon number. Adsorption of straight-chain alcohols is more favorable than that of branched-chain alcohols. Henry's constants increase with increasing number of hydroxyl groups for gas-phase adsorption but decrease for adsorption from aqueous solution due to the strong hydrophilic solvent effect of water. The location of the hydroxyls does not affect significantly the adsorption from aqueous solution but does so in gas-phase adsorption. The saturation pressures for gas-phase adsorption decrease by orders of magnitude from the alcohols to the triols. Nonframework cations increase the adsorption of the small alcohols by an order magnitude at low concentrations (<1 mg/mL), but result in only a factor of 2 increase for larger alcohols like butanol at low concentrations (<0.03 mg/mL), and then decrease the adsorption at higher concentrations. Overall, the simulated results are in reasonable agreement with available experimental data.

Salting-in characteristics of globular proteins.

Protein solubility, and the formation of various solid phases, is of interest in both bioprocessing and the study of protein condensation diseases. Here we examine the the phase behavior of three proteins (chymosin B, ß-lactoglobulin B, and pumpkin seed globulin) previously known to display salting-in behavior, and measure their solubility as a function of pH, ionic strength, and salt type. Although the phase behavior of the three proteins is quantitatively different, general trends emerge. Stable crystal nucleation does not occur within the salting-in region for the proteins examined, despite the crystal being observed as the most stable solid phase. Instead, two types of amorphous phases were found within the salting-in region; additionally, an analog to the instantaneous clouding curve was observed within the salting-in region for chymosin B. Also, protein solutions containing sulfate salts resulted in different crystal morphologies depending on whether Li(2)SO(4) or (NH(4))(2)SO(4) was used.

Interactions and phase behavior of a monoclonal antibody.

Protein phase behavior is implicated in numerous aspects of downstream processing either by design, as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. An improved understanding of protein phase behavior is, therefore, important for developing rational design strategies for important process steps. This work explores the phase behavior of a monoclonal antibody (mAb), IDEC-152, which exhibits liquid-liquid separation, aggregation, gelation, and crystallization. A systematic study of numerous factors, including the effects of solution composition and pH, has been conducted to explore the phase behavior of this antibody. Phenomena observed include a significant dependence of the cloud point on the cation in sulfate salts and nonmonotonic trends in pH dependence. Additionally, conditions for crystallization of this mAb are reported for the first time. Protein-protein interactions, as determined from the osmotic second virial coefficient, are used to interpret the phase behavior.