Solution nonideality related to solute molecular characteristics of amino acids

By measuring the freezing-point depression for dilute, aqueous solutions of all water-soluble amino acids, we test the hypothesis that nonideality in aqueous solutions is due to solute-induced water structuring near hydrophobic surfaces and solute-induced water destructuring in the dipolar electric fields generated by the solute. Nonideality is expressed with a single solute/solvent interaction parameter I, calculated from experimental measure of delta T. A related parameter, I(n), gives a method of directly relating solute characteristics to solute-induced water structuring or destructuring. I(n)-values correlate directly with hydrophobic surface area and inversely with dipolar strength. By comparing the nonideality of amino acids with progressively larger hydrophobic side chains, structuring is shown to increase with hydrophobic surface area at a rate of one perturbed water molecule per 8.8 square angstroms, implying monolayer coverage. Destructuring is attributed to dielectric realignment as described by the Debye-Hückel theory, but with a constant separation of charges in the amino-carboxyl dipole. By using dimers and trimers of glycine and alanine, this destructuring is shown to increase with increasing dipole strength using increased separation of fixed dipolar charges. The capacity to predict nonideal solution behavior on the basis of amino acid characteristics will permit prediction of free energy of transfer to water, which may help predict the energetics of folding and unfolding of proteins based on the characteristics of constituent amino acids.     

The hydrophobic effect. 3. A key ingredient in predicting n-octanol-water partition coefficients

The quantitative development of the mobile order theory in H-bonded liquids is extended to predict the n-octanol/water partition coefficient (P). The log P predictive equation strictly issued from a thermodynamic treatment reduces to a simple linear volume-log P relationship whose intercept and slope encode, respectively, the solvation and entropy effects. For noncomplexing substances, the partition coefficient values result from two volume-dependent entropic contributions reflecting (a) the difference in the exchange entropy between the solute and solvent molecules in the n-octanol and water phases, and (b) the propensity difference between the two H-bonded solvents to induce a hydrophobic effect toward the solute. Although both effects increase, although with opposite signs, compared with the growing molar volume of the partitioned compound, the hydrophobic contribution always predominates favoring the transfer of the solute into the organic phase and hence increasing its partition coefficient. When dealing with complexing chemicals, the hydrophobic effect-related term, though remaining the dominant factor in most cases, is more or less counterbalanced by the formation of H-bonds between the interacting sites of the solute and the n-octanol and water solvent molecules. The log P, corrected for the substantial content of water into n-octanol, is estimated for a number of compounds of environmental and pharmaceutical interest. The extent to which the entropic and enthalpic factors affect the overall partition coefficient value is analyzed.     

13C nuclear magnetic resonance study of molecular motions and conformational transitions in muscle calcium binding parvalbumins

13C nuclear magnetic resonance is used to detect the Ca2+ ion controlled conformational transition in muscle calcium binding parvalbumin and to study its intramolecular motions. Nuclear relaxation parameters are used to evaluate the reorientation rates of the protein and some of the amino acid side chains. While peripheral residues exhibit greater motional freedom than the protein interior, an interesting finding is that significant rapid internal motion is present in the phenylalanine rings comprising the hydrophobic core of the protein.     

Simulating the minimum core for hydrophobic collapse in globular proteins

To investigate the nature of hydrophobic collapse considered to be the driving force in protein folding, we have simulated aqueous solutions of two model hydrophobic solutes, methane and isobutylene. Using a novel methodology for determining contacts, we can precisely follow hydrophobic aggregation as it proceeds through three stages: dispersed, transition, and collapsed. Theoretical modeling of the cluster formation observed by simulation indicates that this aggregation is cooperative and that the simulations favor the formation of a single cluster midway through the transition stage. This defines a minimum solute hydrophobic core volume. We compare this with protein hydrophobic core volumes determined from solved crystal structures. Our analysis shows that the solute core volume roughly estimates the minimum core size required for independent hydrophobic stabilization of a protein and defines a limiting concentration of nonpolar residues that can cause hydrophobic collapse. These results suggest that the physical forces driving aggregation of hydrophobic molecules in water is indeed responsible for protein folding.     

Effect of amino acids on purified rat intestinal brush-border membrane aminooligopeptidase

When a hexapeptide, Leu-Trp-Met-Arg-Phe-Ala, or a pentoapeptide, Leu-Trp-Met-Arg-Phe, was incubated in vitro with a purified aminooligopeptidase from rat small intestinal mucosa, the respective C-terminal dipeptides, Phe-Ala and Arg-Phe, were observed to be resistant to hydrolysis. The resistance of these C-terminal dipeptides to hydrolysis was found to be due mainly to the accumulation of inhibitory hydrophobic amino acids liberated in the incubation mixture. The hydrolysis of various peptides by the brush-border membrane peptidase is inhibited to a varying extent by the hydrophobic amino acids L-tryptophan, L-methionine, L-isoleucine, L-leucine, L-tyrosine, and L-phenylalanine, but not the D-form of these amino acids. The inhibition of the hydrolysis of three dipeptides by hydrophobic amino acids showed these amino acids to be competitive inhibitors (same Vmax, the maximal velocity of the enzyme reaction; different Km, the substrate concentration at which the enzyme reaction is half maximal) of one of the dipeptides while exhibiting a mode of inhibition that was not competitive (different Vmax, different Km) with either of the other two dipeptides. These data indicate that the effect of amino acids on the hydrolytic rate of the brush-border membrane aminooligopeptidases must be considered in studies of intestinal hydrolysis and absorption of peptides.     

Character of Organic Matter in Soil-Aquifer Treatment Systems

The objective of this study was to investigate the character and fate of bulk organics in reclaimed water used for groundwater recharge via soil-aquifer treatment (SAT). The study design followed a watershed guided approach considering hydraulically corresponding samples of drinking water sources, SAT-applied wastewater effluents, and subsequent post-SAT samples representing a series of different travel times in the subsurface. Water samples were fractionated into hydrophobic acids, transphilic acids, and hydrophilic carbon using a XAD resin-based protocol. Extensive characterization of organic carbon in the different samples was performed using state-of-the-art analytical techniques including excitation–emission matrix fluorescence spectroscopy, size exclusion chromatography, carbon-13 nuclear magnetic resonance spectroscopy (13C-NMR), Fourier transform infrared spectroscopy (FTIR), and elemental analysis. During SAT, transphilic and hydrophilic organic matter were preferentially removed. The results generally demonstrated that naturally derived (NOM) and effluent-derived organic matter after SAT overlap extensively in molecular weight distribution, amount and distribution of hydrophobic and hydrophilic carbon fractions, and chemical characteristics based on elemental analysis and 13C-NMR and FTIR spectroscopy. However, the residual portion of the dissolved organic carbon contained both effluent-derived organic matter and NOM.     

Characterization of density gradients prepared by freezing and thawing a sucrose solution

Density gradients of sucrose can be prepared in large numbers by successive freezing and thawing of sucrose solutions. Gradients of other solute molecules, such as salt and detergents, also form and this could affect subsequent sedimentation behavior of some molecules. However, the sedimentation behavior of native and denatured DNA of bacteriophage lambda was essentially isokinetic under the conditions used thus making these gradients comparable with ones prepared manually, at least for preparative sedimentation work with nucleic acids.     

The use of quantitative bacteriologic assessment of bone

A new sub-class of binding protein-dependent transporter with specificity for a broad range of polar amino acids has been identified by sequence comparison, in Rhizobium leguminosarum, Rhodobacter capsulatus, Escherichia coli and Pseudomonas fluorescens. Southern blotting and PCR analysis has shown that transporters from this new sub-class are widely distributed in Gram-negative bacteria, including, in addition to the above, Citrobacter freundii, Erwinia carotovorum and Rhizobium meliloti. ABC transporters of polar amino acids can be divided into two groups: those with narrow solute specificity and the newly identified sub-class with broad solute specificity. The binding and inner membrane proteins from transporters with a broad solute specificity are larger by approximately 30% than those with a narrow solute specificity. Multiple alignment of the inner membrane proteins from all sequenced polar amino acid transporters indicates there is an N-terminal conserved region that may be involved in solute specificity. A conserved arginine or lysine at residue 30 of this region is changed to glutamate in arginine transporters. Residue 53 also has a strong correlation with the charge on the transported solute, with basic amino acid transporters replacing an aliphatic amino acid at this position with a negatively charged amino acid. The general amino acid permease from R. leguminosarum, which will transport aliphatic as well as basic and acidic amino acids, juxtaposes two prolines at residues 52 and 53 of the N-terminal conserved region.     

Statistical analysis of predicted transmembrane alpha-helices

Statistical analyses were undertaken for putative transmembrane alpha-helices obtained from a database representing the subset of membrane proteins available in Swiss-Prot. The average length of a transmembrane alpha-helix was found to be 22-21 amino acids with a large variation around the mean. The transfer free energy from water to oil of a transmembrane alpha-helix in bitopic proteins, -48 kcal/mol, is higher than that in polytopic proteins, -39 kcal/mol, and is nearly identical to that obtained by assuming a random distribution of solely hydrophobic amino acids in the alpha-helix. The amino acid composition of hydrophobic residues is similar in bitopic and polytopic proteins. In contrast, the more polar the amino acids are, the less likely they are to be found in bitopic proteins compared to polytopic ones. This most likely reflects the ability of alpha-helical bundles to shield the polarity of residues from the hydrophobic bilayer. One half of all amino acids were distributed nonrandomly in both bitopic and polytopic proteins. A preference was found for tyrosine and tryptophan residues to be at the ends of transmembrane alpha-helices. Correlated distribution analysis of amino acid pairs indicated that most amino acids are independently distributed in each helix. Exceptions are cysteine, tyrosine, and tryptophan which appear to cluster closely to one another and glycines which are preferentially found on the same side of alpha-helices.     

Solvent relaxation by uniformly magnetized solute spheres. The classical-quantal connection

RATIONALE AND OBJECTIVES: Large magnetic entities, with diameters in the range of 4 nm to 4 microns, are becoming of increasing interest for magnetic resonance imaging (MRI). The smaller are iron oxide nanoparticles, used for the RE system, and the larger are deoxygenated blood cells, for functional MRI. It can be useful to model such systems as magnetized solute spheres in water. Classical computations of 1/T2 have been reported for the larger particles, in the micron range, where the computational complexities are simplified by Monte Carlo methods. For smaller particles, the quantum mechanical (quantal) expressions for outer sphere relaxation, for both 1/T1 and 1/T2, have been available for some time, and are particularly simple to apply at MRI fields. The questions that arise, and which the author addresses, are how to interrelate the classical and quantal approaches and when to use which. METHODS: The author compares published results of Monte Carlo calculations of 1/T2 for diamagnetic polystyrene solute spheres of various sizes in water, made paramagnetic by addition of dysprosium-(DTPA)2-, with quantum mechanical outer sphere theory applied to the same system. The latter includes the usual assumption of motional narrowing and yields both 1/T1 and 1/T2. RESULTS: For particles with diameters less than about 1 micron, both approaches give identical results for 1/T2. For larger particles, the conditions for motional narrowing breakdown, and quantal theory overestimates 1/T2. In addition, in the particular system studied, relaxation becomes so effective near solute that there is insufficient time for all water molecules to experience their maximal effect. Classical theory handles this well whereas quantal theory does not. CONCLUSIONS: In comparing the classical and quantal approaches, one balances computational complexity but broader applicability with more limited but far simpler mathematics. In addition, because the quantal approach shows that 1/T1 and 1/T2 are intimately related, the author suggests, by analogy, how to extend classical methods to computation of 1/T1.     

HSA-solute interactions, enantioselectivity, and binding site geometrical characteristics

Recently, a theoretical model was proposed to study the existence of pockets of acetonitrile (ACN) called clusters in a hydroorganic mixture. The proposal used ACN as an interaction organic modifier between D,L-dansyl amino acids and their binding site in human serum albumin at site II. This solute binding is governed by primary and secondary interactions. The primary interactions are under the dependence of the solute solvation by ACN clusters and electrostatic interactions. Following this first step, the solute engages strong short-range interactions with the residues of site II. Using a biochromatographic approach, the solute binding, i.e., the solute retention, was divided into two dielectric constant (epsilon) ranges. In the first range, epsilon > epsilon c (epsilon c is the critical dielectric constant); the primary and secondary nonstereoselective electrostatic interactions were the major contributions to the variation in the solute binding with the ACN fraction in the mixture. In the second range, epsilon < epsilon c, the solute retention variation with the ACN fraction was governed by its solvation by the ACN clusters and also by the secondary hydrophobic stereoselective interaction. The mathematical model developed provided the determination of the surface charge density of site II as well as the cluster number that solvates each solute.     

Side-chains in native and random coil protein conformations. Analysis of NMR coupling constants and chi1 torsion angle preferences

The behaviour of amino acid side-chains in proteins in solution has been characterised by analysing NMR 3JHalphaH beta coupling constants and crystallographic chi1 torsion angles. Side-chains both in the core of native folded proteins and in situations where there is an absence of close packing including the random coil state have been considered. An analysis of experimental 3JHalphaH beta coupling constant data for ten proteins shows that in the core of native proteins a very close similarity is observed between the chi1 conformations adopted in solution and in crystals. There is clear evidence, however, for significant motional averaging about the chi1 torsion angles in solution. Using a model of a Gaussian distribution about the average torsion angles the extent of these fluctuations has been quantified; the standard deviation for the motion is 26 degrees, the fluctuations about chi1 in the protein core being similar in size to those found for main-chain phi torsion angles in solution. From the distribution of chi1 torsion angles in a data base of protein crystal structures, torsion angle populations and coupling constants have been predicted for a random coil polypeptide. Significant variations in the chi1 distributions for different amino acids give differences in the predicted coupling constants; for 3JHalphaH beta, for example, values of 5.1 and 5.7 Hz are predicted for serine compared with 4.9 and 9.9 Hz for leucine. Experimental data for short unstructured peptides show an excellent agreement with the predictions, indicating that the overall chi1 distributions in protein crystals reflect the local preferences of the amino acids. Predictions from the protein data base therefore provide an important framework for interpreting experimental data for non-native protein conformations and for residues on the surface of folded proteins.     

Structure and expression of human fibroblast growth factor-10

We isolated the cDNA encoding a novel member of the human fibroblast growth factor (FGF) family from the lung. The cDNA encodes a protein of 208 amino acids with high sequence homology (95.6%) to rat FGF-10, indicating that the protein is human FGF-10. Human FGF-10 as well as rat FGF-10 has a hydrophobic amino terminus ( approximately 40 amino acids), which may serve as a signal sequence. The apparent evolutionary relationships of human FGFs indicate that FGF-10 is closest to FGF-7. Chromosomal localization of the human FGF-10 gene was examined by in situ hybridization. The gene was found to map to the 5p12-p13 region. Human FGF-10 (amino acids 40 to 208 with a methionine residue at the amino terminus) was produced in Escherichia coli and purified from the cell lysate. Recombinant human FGF-10 (approximately 19 kDa) showed mitogenic activity for fetal rat keratinizing epidermal cells, but essentially no activity for NIH/3T3 cells, fibroblasts. The specificity of mitogenic activity of FGF-10 is similar to that of FGF-7 but distinct from that of bFGF. In structure and biological activity, FGF-10 is similar to FGF-7.     

Protein rotational relaxation as studied by solvent 1H and 2H magnetic relaxation

Earlier studies of the magnetic field dependence of the nuclear spin magnetic relaxation rate of solvent protons in solutions of diamagnetic proteins have indicated that this dependence (called relaxation dispersion) is related to the rotational Brownian motion of solute proteins. In essence, the dispersion is such that 1/T1 (the proton spin-lattice relaxation rate) decreases monotonically as the magnetic field is increased from a very low value (approximately 10 Oe); the dispersion has a point of inflection at a value of magnetic field which depends on protein size, shape, concentration, temperature, and solvent composition. The value of the proton Larmor precession frequency nu(c) at the inflection field appears to relate to tau (R), the rotational relaxation time of the protein molecules. We have measured proton relaxation dispersions for solutions of various proteins that span a three-decade range of molecular weights, and for one sample of transfer ribonucleic acid. We have also measured deuteron relaxation dispersions for solutions of three proteins: lysozyme, carbonmonoxyhemoglobin, and Helix pomatia hemocyanin with molecular weight 900 000. A quantitative relationship between both proton and deuteron dispersion data and protein rotational relaxation is confirmed, and the point is made that magnetic dispersion measurements are of very general applicability for measuring the rotational relaxation rate of macromolecules in solution. It has been previously shown that the influence of proton motion on the relaxation behavior of the solvent is not due to exchange of solvent molecules between the bulk solvent and a hydration region of the protein. In the present paper, we suggest that the interaction results from a long range hydrodynamic effect fundamental to the situation of large Brownian particles in an essentially continuum fluid. The general features of the proposed mechanism are indicated, but no theoretical computations are presented.     

Molecular characterization of a broad selectivity neutral solute channel

In all living cells, coordination of solute and water movement across cell membranes is of critical importance for osmotic balance. The current concept is that these processes are of distinct biophysical nature. Here we report the expression cloning of a liver cDNA encoding a unique promiscuous solute channel (AQP9) that confers high permeability for both solutes and water. AQP9 mediates passage of a wide variety of non-charged solutes including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercury-sensitive manner, whereas amino acids, cyclic sugars, Na+, K+, Cl-, and deprotonated monocarboxylates are excluded. The properties of AQP9 define a new evolutionary branch of the major intrinsic protein family of aquaporin proteins and describe a previously unknown mechanism by which a large variety of solutes and water can pass through a single pore, enabling rapid cellular uptake or exit of metabolites with minimal osmotic perturbation.     

A two-phase model for controlled drug release from biphasic polymer hydrogels

A comprehensive two phase model is developed to describe the sustained release of a solute or drug from a biphasic hydrogel substrate. Such a material consists of a continuous hydrophilic phase (polymer backbone in water) and a dispersion of spherical microdomains made of the hydrophobic side chains of the polymer organised in a micelle like fashion. The solute or drug is assumed to be encapsulated within the dispersed microdomains, and to diffuse from the interior to the surface of the microdomain where it exchanges following a Langmuir isotherm. Mass transfer to the bulk phase occurs by desorption of the drug from the surface through a driving force that is proportional to the difference of surface and bulk concentration. Accordingly the drug is released to the surroundings by diffusion through the bulk. Depending on the values of the Langmuir constant and assuming well stirred behaviour in the interior of the microdomain, the present model results in either of the two asymptotic models developed in previous studies. The results of a parametric study show that the desired steady state flux of a specific drug to the surroundings may be obtained given appropriate values of structural properties of the material. This conclusion is further supported when using this model to simulate earlier experimental results. The polymer structural properties can be manipulated easily during the fabrication of dispersed-phase networks, as indicated by preliminary experiments.     

Biological water and its role in the effects of alcohol

Alcohol and water compete with each other on target membrane molecules, specifically, lipids and proteins near the membrane surface. The basis for this competition is the hydrogen bonding capability of both compounds. But alcohol's amphiphilic properties give it the capability to be attracted simultaneously to both hydrophobic and hydrophilic targets. Thus, alcohol could bind certain targets preferentially and displace water, leading to conformational consequences. This article reviews the clustering and organized character of biological water, which modulates the conformation of membrane surface molecules, particularly receptor protein. Any alcohol-induced displacement of biological water on or inside of membrane proteins creates the opportunity for allosteric change in membrane receptors. This interaction may also prevail in organelles, such as the Golgi apparatus, which have relatively low concentrations of bulk water. Target molecules of particular interest in neuronal membrane are zwitteronic phospholipids, gangliosides, and membrane proteins, including glycoproteins. FTIR and NMR spectroscopic evidence from model membrane systems shows that alcohol has a nonstereospecific binding capability for membrane surface molecules and that such binding occurs at sites that are otherwise occupied by hydrogen-bonded water. The significance of these effects seems to lie in the need to learn more about biological water as an active participant in biochemical actions. Proposed herein is a new working hypothesis that the molecular targets of ethanol action most deserving of study are those where water is trapped and there is little bulk water. Proteins (enzymes and receptors) certainly differ in this regard, as do organelles.     

Polynucleotide hydrolysis by pancreatic ribonuclease immobilized on carboxymethylcellulose]

Analysis of snail-conditioned water (SCW) from Helisoma trivolvis revealed 17 free amino acids. Those in great concentration were glycine, serine, and alanine. The concentration of sialic acid was found to be twice that of the most abundant amino acid. The behavior of miracidia of Megalodiscus temperatus, measured by the contact with return method, to agar cylinders containing single amino acids and sialic acid indicated greater responses to polar molecules charged either positively or negatively at neutral pH. The molecules elicting the greatest response were aspartic, glutamic, and sialic acid. No correlation was found between concentration of amino acids in H. trivolvis SCW and response of M. temperatus miracidia.     

Hydration and conformational equilibria of simple hydrophobic and amphiphilic solutes

We consider whether the continuum model of hydration optimized to reproduce vacuum-to-water transfer free energies simultaneously describes the hydration free energy contributions to conformational equilibria of the same solutes in water. To this end, transfer and conformational free energies of idealized hydrophobic and amphiphilic solutes in water are calculated from explicit water simulations and compared to continuum model predictions. As benchmark hydrophobic solutes, we examine the hydration of linear alkanes from methane through hexane. Amphiphilic solutes were created by adding a charge of +/-1e to a terminal methyl group of butane. We find that phenomenological continuum parameters fit to transfer free energies are significantly different from those fit to conformational free energies of our model solutes. This difference is attributed to continuum model parameters that depend on solute conformation in water, and leads to effective values for the free energy/surface area coefficient and Born radii that best describe conformational equilibrium. In light of these results, we believe that continuum models of hydration optimized to fit transfer free energies do not accurately capture the balance between hydrophobic and electrostatic contributions that determines the solute conformational state in aqueous solution.