Solvophobic forces and molecular surface area changes in drug-biomolecule associations as with actinomycin-deoxyguanosine in a wide range of methanol/water mixtures

A method is given to predict the unitary free energies of complexation between drug-like and nucleoside-like biomolecules in a range of mixed solvent compositions. A stability maximum for the actinomycin (A)-deoxyguanosine (D) complex at 8% MeOH (v/v) in methanol/water mixtures is correctly predicted by the theory in agreement with existing experimental data. The molecular surface areas of A and D exposed to the solvent are found to diminish by 36.4 A(2) upon association. The 'microthermodynamic differential surface tension' of the solvophobic theory obtained for nucleoside-like and organic molecules in contact with MeOH/H2O can be used to predict the solvent effect free energies in other such molecular or biopolymeric associations in solution.     

ZL-DHP lignin model compound at the air-water interface

In this paper we present our surface chemistry studies of enzymatically polymerized, poly-coniferyl alcohol lignin model compound (dehydrogenate polymer a.k.a. ZL-DHP) at the air-water interface. Using the CHCl(3)/MeOH (5:1 v/v) spreading solvent, we found an average molecular area of ZL-DHP of approximately 1200 A(2). The monolayer expresses a high compressibility with a collapsed area of 500 A(2) and collapsed surface pressure of 28 mN m(-1). In the range of applied surface pressures, ZL-DHP polymer have no phase changes, as shown by the very high linearity (R=0.994) of absorbance vs. surface pressure cure. There was no symmetry transitions observed as shown by absence of shifts of absorption peak maximums.     

Partitioning of tryptophan side-chain analogs between water and cyclohexane.

We have measured the partitioning of the tryptophan side-chain analogs 3-methylindole and N-methylindole between water and cyclohexane over the temperature range 8-55 degrees C to investigate the relative contribution of the imine-NH- to the free energy of transfer. We take advantage of the fact that the indole imine nitrogen is blocked by a methyl group in N-methylindole. Unlike previous studies, we take into account the water present in the cyclohexane phase. Free energies of partitioning were calculated using mole-fraction, volume-fraction, and Flory-Huggins-corrected volume-fraction partition coefficients [De Young, L. R., & Dill, K. A. (1990) J. Phys. Chem. 94, 801-809; Sharp, K. A., Nicholls, A., Friedman, R., & Honig, B. (1991) Biochemistry 30, 9686-9697]. These approaches account for configurational entropy changes in different ways and thus lead to different values for the calculated free energies of transfer. There is a 2-3-fold difference in the free energies calculated from our measurements, using the different units. Independent of units, the partitioning of both compounds involves identical entropy changes. However, 3-methylindole has an additional unfavorable enthalpic contribution to partitioning into cyclohexane of +1.6 kcal/mol (independent of units) which is presumably the cost of removing the indole -NH- group from water and transferring it to cyclohexane. In cyclohexane, 3-methylindole forms hydrogen bonds with water that cause water to copartition into cyclohexane with the solute. A method is described which allows the partitioning process to be examined independent of subsequent interactions with water in the solvent.     

Use of a Novel Sub‐2 µm Silica Hydride Vancomycin Stationary Phase in Nano‐Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano‐liquid chromatography (nano‐LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns‐AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74–4.17 and 1.39–1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41–6.09 and α 1.28–2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. Chirality 27:767–772, 2015. © 2015 Wiley Periodicals, Inc.     

Pseudo-dynamic contact surface areas: estimation of apolar bonding

A new Monte Carlo based algorithm has been written for the computation of pseudo-dynamic contact surface areas. The linear correlation of this contact area with solute transfer free energies (water leads to organic liquid) is established for apolar amino acid side chains. The slope of these linear plots, deltaGosp, is a unitary free energy which has potential use in the estimation of apolar bond free energies in proteins. The magnitude of deltaGosp is dependent upon the nature of the organic solvent involved in the transfer process, varying from 86 to 130 cal/A2. Analogues linear correlations with the same range of deltaGosp values are observed for inhibitors of protein-catalyzed reactions.     

Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface

Two thermodynamic models have been developed to interpret the preferential accumulation or exclusion of solutes in the vicinity of biopolymer surface and the effects of these solutes on protein processes. The local-bulk partitioning model treats solute (and water) as partitioning between the region at/or near the protein surface (the local domain) and the bulk solution. The solvent exchange model analyzes a 1:1 competition between water and solute molecules for independent surface sites. Here we apply each of these models to interpret thermodynamic data for the interactions of urea and the osmoprotectant glycine betaine (N,N,N-trimethylglycine; GB) with the surface exposed in unfolding the marginally stable lacI HTH DNA binding domain. The partition coefficient K(P) quantifying accumulation of urea at this protein surface (K(P) approximately equal 1.1) is only weakly dependent on urea concentration up to 6 M urea. However, K(P) quantifying exclusion of GB from the vicinity of this protein surface increases from 0.83 (extrapolated to 0 M GB) to 1.0 (indicating that local and bulk GB concentrations are equal) at 4 M GB (activity > 40 M). We interpret the significant concentration dependence of K(P) for GB, predicted to be general for excluded, nonideal solutes such as GB, as a modest (8%) attenuation of the GB concentration dependence of solute nonideality in the local domain relative to that in the bulk solution. Above 4 M, K(P) for the interaction of GB with the surface exposed in protein unfolding is predicted to exceed unity, which explains the maximum in thermal stability observed for RNase and lysozyme at 4 M GB (Santoro, M. M., Liu, Y. F., Khan, S. M. A., Hou, L. X., and Bolen, D. W. (1992) Biochemistry 31, 5278-5283). Both thermodynamic models provide good two-parameter fits to GB and urea data for lacI HTH unfolding over a wide concentration range. The solute partitioning model allows for a full spectrum of attenuation effects in the local domain, encompasses the cases treated by the competitive binding model, and provides a somewhat better two-parameter fit of effects of high GB concentration on lacI HTH stability. Parameters of this fit should be applicable to isothermal and thermal unfolding data for all proteins with similar compositions of surface exposed in unfolding.     

Denaturation of proteins in methanol/water mixtures

The solvophobic theory developed earlier by Sinanoglu introducing the use of molecular surface areas and microthermodynamic surface and interfacial tensions at molecular dimensions is applied to the interpretation of calorimetric data on denaturation of lysozyme in a wide range of methanol/water mixtures. The experimental values of standard unitary free energies of denaturation correlate well with our predictions. The molecular surface area change of the protein upon denaturation is evaluated using the solvophobic theory. The maximum in the stability of the native form of the protein is predicted to occur at 8% (v/v) methanol. This is found to be in agreement with the experimental results.     

Interaction of gangliosides with proteins depending on oligosaccharide chain and protein surface modification.

By using neutron and synchrotron x-ray small-angle scattering techniques, we investigated the process of the complexation of gangliosides with proteins. We treated monosialoganglioside (G(M1)), disialoganglioside (G(D1a)), and a mixture of G(M1)/G(D1a). Proteins used were bovine serum albumins whose surfaces were modified with different sugars (deoxy-D-galactose, deoxy-L-fucose, deoxymaltitol, and deoxycellobiitol), which were used as model glycoproteins in a membrane. We found that the complexation of gangliosides with albumins greatly depends on the combination of ganglioside species and protein surface modification. With a varying protein/ganglioside ratio in a buffer solution at pH 7, the complexation of G(M1) or G(D1a) with albumins modified by monosaccharides appears to be less destructive for ganglioside aggregate structures in forming large complexes; the complexation of G(D1a) with the albumins modified by disaccharides induces the formation of complexes with a dimeric structure; and the complexation of G(M1) with albumins modified by disaccharides, to form small complexes, is very destructive. The present results show a strong dependence of the interaction between ganglioside and protein on the characteristics of the ganglioside and protein surface, which would relate to a physiological function of gangliosides, such as a function regulating the receptor activity of glycoproteins in a cell membrane.     

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.     

Diffusion and saponification inside porous cellulose triacetate fibers

Cellulose triacetate (CTA) fibers were partially hydrolyzed in 0.054 N solutions of NaOH/H(2)O and NaOMe/MeOH. The surface concentration of acetyl groups was determined using ATR-FTIR. Total acetyl content was determined by the alkaline hydrolysis method. Fiber cross-sections were stained with Congo red in order to examine the interface between reacted and unreacted material; these data were used to estimate the rate constant k and effective diffusivity D(B) for each reagent during the early stages of reaction by means of a volume-based unreacted core model. For NaOH/H(2)O, k = 0.37 L mol(-1) min(-1) and D(B) = 6.2 x 10(-7) cm(2)/sec; for NaOMe/MeOH, k = 4.0 L mol(-1) min(-1) and D(B) = 5.7 x 10(-6) cm(2)/sec. The NaOMe/MeOH reaction has a larger rate constant due to solvent effects and the greater nucleophilicity of MeO(-) as compared to OH(-); the reaction has a larger effective diffusivity because CTA swells more in MeOH than it does in water. Similarities between calculated concentration profiles for each case indicate that the relatively diffuse interface seen in fibers from the NaOMe/MeOH reaction results from factors not considered in the model; shrinkage of stained fiber cross-sections suggests that increased disruption of intermolecular forces may be the cause.     

ESI-MS investigation of solvent effects on the chiral recognition capacity of tartar emetic towards neutral side-chain amino acids

The effect of solvent systems on previously-reported ESI-MS based proton-assisted enantioselective molecular recognition phenomena of tartar emetic, L-antimony(III)-tartrate, was evaluated. This was achieved by carrying out a series of competitive binding experiments using chiral selectors, bis(sodium) D- and -L-antimony(III)-tartrates with chiral selectands, neutral side-chain amino acid enantiomeric isotopomers of alanine (Ala), valine (Val), leucine (Leu) and phenylalanine (Phe), in three different solvent systems, ACN/H(2)O (75/25 v/v), H(2)O (100%) and H(2)O/MeOH (25/75 v/v). Observations from these experiments suggest that the effect of solvent systems on previously reported proton-assisted chiral recognition capacity of D,L-antimony(III)-tartrates is small, but not negligible. It was observed that an ACN/H(2)O (75/25 v/v) solvent system facilitates and enhances the chiral discrimination capacity of protonated {[D,L-Sb(2)-tar(2)][H]}(-) ionic species. Further, amino acid enantiomers showed a general trend of increasing selectivity order, Val ≤ Ala < Leu ≈ Phe towards the protonated {[D,L-Sb(2)-tar(2)][H]}(-) ionic species which was independent of the solvent system employed. The lack of enantioselective binding for {[D,L-Sb(2)-tar(2)]}(2-) ionic species was consistently recorded in respective mass spectra from all performed experiments, which suggests that ESI-friendly solvent systems have no effect and do not influence this phenomenon.     

High-yield purification of lung surfactant proteins sp-b and sp-c and the effects on surface activity

Several protocols for purification of milligram quantities of lung surfactant proteins (SP)-B and SP-C were studied for separation efficiency and surface activity of the isolated proteins recombined with synthetic phospholipids (SPL). SP-B and SP-C were obtained from calf lung surfactant extract by C8 chromatography with isocratic elution by either of three solvent systems: 7:1:0.4 MeOH/CHCl(3)/5% 0.1 M HCl (solvent A), 7:1 MeOH/CHCl(3)+ 0.1% TFA (solvent B), and 7:1:0.4 MeOH/CHCl(3)/H(2)O + 0.1% TFA (solvent C). Solvents A and C yielded pure apoprotein in a single pass, with estimated total protein recoveries of >85 and >90%, respectively. Solvent B was less effective in purifying SP-B and SP-C, had a lower recovery efficiency, and gave isolates with less surface activity. Mixtures of SPL plus SP-B eluted with solvents A and C adsorbed to equilibrium surface tensions of 21-22 mN/m and reached minimum surface tensions <1 mN/m during dynamic cycling. Mixtures of SPL with SP-C obtained with solvents A and C had equilibrium surface tensions of 26-27 mN/m and minimum dynamic values of 2-7 mN/m. The ability to obtain milligrams of virtually lipid-free SP-B and SP-C in a single column pass will facilitate research on their biological, structural, and biophysical properties.     

Electro-optical studies on the electric field-induced helix-to-coil transition of poly(l-ornithine hydrobromide) in methanol/water mixtures

Transient electric birefringence and circular dichroism measurements have been made on poly(L-ornithine hydrobromide) in methanol/water mixtures of various compositions. The specific Kerr constant and the molar ellipticity at 222 nm underwent an abrupt change between 93 and 98% (v/v) methanol at 25°C corresponding to a solvent-induced helix-coil transition. Anomalous birefringence transients were observed at high electric fields between 96 and 98% (v/v) methanol, i.e. on the helix side of the transition region. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different solvent compositions could be superimposed on one another by horizontal and vertical shifts, except for the range where anomalous birefringence transient were observed. This behavior served to determine the threshold field strength. The results indicate that a conformational change from the charged helix to the charged coils is induced by high electric fields in this system, as in the cases of poly(L-lysine hydrobromide) and poly(L-αγ-diaminobutyric acid hydrochloride) in methanol/water mixtures.     

Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix

Heteronuclear 3D and 4D NMR experiments have been used to obtain 1H, 13C, and 15N backbone chemical shift assignments in Ca(2+)-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca(2+)-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca(2+)-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (kexch greater than 10 s-1 at pH 7, 35 degrees C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.     

Extending the diffuse layer model of surface acidity behaviour: III. Estimating bound site activity coefficients

Abstract

Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in application these terms are ignored based on one or more of the following assumptions: (1) bound site activity coefficients cancel out in the mass action quotients; (2) bound sites display ideal behaviour; and/or (3) these energies are already included in the exponential Boltzmann terms. In this work it is demonstrated that the bound site charging energy terms discussed in the two previous papers in this series have both conceptual and computational analogies to the charging energy contribution to the activity coefficients obtained from the Debye–Huckel Limiting Law. On high charge density colloidal particles at constant counterion condensation (τ), these charging energies can be related to the surface potential (ψ) by: ΔG_charging = (1 – τ)Fψ (where F is the Faraday constant). If one assumes a maximum practical accuracy of ± 10% in experimental estimates of ψ, then it is suggested that charging energies are likely to be experimentally indiscernible under conditions where τ > 0.9. These findings support the historical practice of ignoring bound site activity coefficients with the 2-pK diffuse layer surface complexation model in the following situations: for spherical particles with a radius ≥ 0.1 μm at ionic strengths ≥ 0.001 M (1 : 1), and for spherical particles with a radius >0.01 μm at an ionic strength >0.1 M (1 : 1). In contrast, charging energies (and non-ideal behaviour) are predicted to be significant at all charge densities and ionic strengths for spherical particles with a radius of 0.001 μm.     

Dehydration of a polyether type extraction agent and of the corresponding K+ complex: insights into liquid-liquid extraction mechanisms by quantum chemical methods

In this paper we report a quantum chemical study performed at the B3LYP/6-311G++(d,p) level of theory on structural and energetic aspects of the sequential dehydration of a tetra-hydrated polyethylene-glycol type podand (1,2-bis-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-benzene, hereafter b33) and its complex with the K(+) cation. Thermodynamical parameters were determined by hessian quantum calculations performed using a self-consistent reaction field (SCRF) method, taking into account solvent (dichloromethane) effects. The results allowed the estimation of dehydration enthalpies, entropies and free energies for the hydrated free b33 podand and its corresponding K(+) cation complex in dichloromethane. The low absolute values found for the dehydration free energies as well as the structural features found for the optimized structures and the corresponding basis superposition calculated interaction energies, support the hypothesis of an interfacial complexation type mechanism governing the assisted extraction of K(+) from an aqueous toward an organic phase, in liquid/liquid extraction.     

pH dependence of stability of the 10th human fibronectin type III domain: a computational study

We present detailed computational studies based on electrostatic calculations to evaluate the origins of pKa values and the pH dependence of stability for the 10th type III domain of human fibronectin (FNfn10). One of our goals is to validate the calculation protocols by comparison to experimental data (Koide, A.; Jordan, M. R.; Horner, S.; Batori, V.; Koide, S. Biochemistry 2001, 40, 10326-10333). Another goal is to evaluate the sensitivity of the calculated ionization free energies and apparent pKa values on local structural fluctuations, which do not alter the structural convergence to a particular architecture, by using a complete ensemble of solution NMR structures and the NMR average minimized structure of FNfn10 (Main, A. L.; Harvey, T. S.; Baron, M.; Boyd, J.; Campbell, I. D. Cell 1992, 71, 671-678). Our calculations demonstrate that, at high ionic strength, FNfn10 is more stable at low pH compared to neutral pH, in overall agreement with experimental data. This behavior is attributed to contributions from unfavorable Coulombic interactions in a surface patch for the pairs Asp7-Glu9 and Asp7-Asp23. The unfavorable interactions are decreased at low pH, where the acidic residues become neutral, and are further decreased at high ionic strength because of increased screening by salt ions. Elimination of the unfavorable interactions in the theoretical mutants Asp7Asn (D7N) and Asp7Lys (D7K) produce higher calculated stabilities at neutral pH and any ionic strength compared to the wild-type, in agreement with the experimental data. We also discuss subtleties in the calculated apparent pKa values and ionization free energies, which are not in agreement with the experimental data. This work demonstrates that comparative electrostatic calculations can provide rapid predictions of pH-dependent properties of proteins and can be significant aids in guiding the design of proteins with tailored properties.     

Hydrophobicity of the peptide C=O...H-N hydrogen-bonded group

The hydrophobicity of the peptide C=O ... H-N hydrogen-bonded group is an important parameter that determines the structure of proteins in water and in biological membranes, and therefore the free energy of transferring this group from water to non-polar solvents should be determined accurately. The essential work on this problem was carried out by Klotz and co-workers, and has been summarized elsewhere. Using N-methylacetamide as a model peptide, the free energies of the following processes were determined; (1) formation of the C=O ... H-N bond in water, (2) formation of the C=O ... N-N bond in CCl4, and (3) transfer of N-methylacetamide from water to CCl4. (4) From (3), the free energy of transferring the non-hydrogen bonded (C=O, H-N) group from water to CCl4 was calculated. When the free energies of (1), (2) and (4) are combined, one finds that the free energy of transferring the C=O ... H-N group from water to CCl4 is a surprising -1.4 kcal/mol (1 cal = 4.184 J). This number does not seem reasonable, since it implies that the C=O ... H-N group is about as hydrophobic as an isopropyl group, i.e. the side-chain of valine. In the present report, it is shown that this apparent hydrophobicity results from an underestimation of the free energy contribution that the methyl groups make to the transfer of N-methylacetamide from water to CCl4. When appropriate methyl group transfer free energies are used, one finds that the free energy of transferring the C=O ... H-N group from water to CCl4 is +0.62 kcal/mol. Therefore, this group is relatively insensitive to solvent polarity. A similar calculation shows that the free energy of transferring the C=O ... H-O hydrogen-bonded group from water to benzene is +0.55 kcal/mol.     

The solvation contribution to the binding energy of DNA with non-intercalating antibiotics.

The influence of the solvent on the binding energies to DNA of six non-intercalating antibiotics - netropsin, distamycin-3, distamycin-2, SN 18071, berenil and stilbamidine - is evaluated by combining the effect of the first hydration shell with that of bulk water. The first effect is computed by a methodology based on a spherical/point dipole model of water and limited to electrostatic interaction energies. Hydration shells are obtained which are energy optimized with respect to both water-solute and water-water interactions for the complexes and for the isolated DNA oligomers and ligands. The method allows even very large complexes to be studied in reasonable computation times. The second effect is introduced via a cavity treatment. It is shown that if the vacuum interaction energies already predict correctly the preference of the ligands for the minor groove of AT sequences of B-DNA, the introduction of the solvation effect is indispensable for reproducing the order of affinity of the ligands and for bringing the values of the complexation energies into close agreement with experimental data.