Watching small molecules move: Interrogating ionic channels using neutral solutes

Whether they are small enough to wriggle through the current-carrying part of an ionic channel or big enough to be kept outside and thus able to exert an osmotic stress on the channel space, polymers interact with channels in several instructive ways. The osmotic stress of excluded polymers allows one to measure the number of water molecules that come out of the channel in transitions between various open to closed states. The loss of osmotic activity, due to the partial or completely unrestricted admission of small polymers becomes a measure of the transfer probabilities of polymers from solution to small cavities; it provides an opportunity to study polymer conformation in a perfectly sieved preparation. Current fluctuations due to the partial blockage by a transient polymer are converted into estimates of times of passage and diffusion constants of polymers in channels. These estimates show how a channel whose functional states last for milliseconds is able to average over the interactions with polymers, interactions that last only microseconds. One sees clearly that in this averaging, the macromolecular channel is large enough to react like a macroscopic object to the chemical potentials of the species that modulate its activity.     

Exclusion of alcohols from spermidine-DNA assemblies: probing the physical basis of preferential hydration

The interaction of the alcohols 2-methyl-2,4-pentanediol (MPD) and 2-propanol and of glycerol with condensed spermidine(3+)-DNA arrays are investigated with direct force measurements using osmotic stress coupled with X-ray scattering. Thermodynamic forces between DNA helices are measured from the dependence of helical interaxial spacings on the osmotic pressure applied by poly(ethylene glycol) solutions in equilibrium with the DNA phase. The sensitivity of these forces to solute concentration can be transformed into a change in the number of excess or deficit solutes or waters in the DNA phase by applying the Gibbs-Duhem equation. The alcohols examined are excluded from the condensed DNA array and strongly affect the osmotic stress force curves. DNA is preferentially hydrated. MPD is significantly more excluded than 2-propanol. The exclusion of these alcohols, however, is not due to a steric repulsion since glycerol that is intermediate in size between MPD and 2-propanol does not observably affect DNA force curves. As the distance between DNA helices varies, the change in the number of excess waters is independent of alcohol concentration for each alcohol. These solutes are acting osmotically on the condensed array. The distance dependence of exclusion indicates that repulsive water structuring forces dominate the interaction of alcohols with the DNA surface. The exclusion measured for these condensed arrays can quantitatively account for the effect of these alcohols on the precipitation of DNA from dilute solution by spermidine(3+).     

Solute inaccessible aqueous volume changes during opening of the potassium channel of the squid giant axon.

We have applied solutions with varying osmotic pressures symmetrically to the inside and outside of perfused, TTX-treated, giant axons. The potassium conductance G decreased with increasing osmotic stress, but there was no effect on either the shape or the position of the voltage-current curve. One must distinguish three possible actions of the osmotic agent: osmotic stress, channel blocking, and lowered solution conductivity. To do so, we compared results obtained working with pairs of internal and external solutions of either (a) equal osmotic stress, (b) equal conductivity, or (c) the same blocking agent. There was the same change in G irrespective of the type of stressing species (sorbitol or sucrose); this provides some evidence against a blocking mechanism. The conductivity of the external solution had a small effect on K currents; internal solution conductivity had none. A change in series resistance of the Schwann cell layer could account for the small effect of external solution conductivity. The primary cause of G depression appears, then, to be the applied osmotic stress. Using this result, we have developed models in which the channel has a transition between closed states under voltage control but osmotically insensitive and a closed/open step that is voltage-independent but osmotically sensitive. We have assumed that the conductance of this open state does not change with osmotic stress. In this way, we estimate that an additional 1,350 +/- 200 A3 or 40-50 molecules of solute-inaccessible water appear to associate with the average delayed rectifier potassium channel of the squid axon when it opens.     

Mechanism of cryoprotection by extracellular polymeric solutes.

To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits.     

Exclusion of maltodextrins from phosphatidylcholine multilayers during dehydration: effects on membrane phase behaviour

The effect of increasing solute size on phosphatidylcholine phase behaviour at a range of hydrations was investigated using differential scanning calorimetry. Dehydration of phospholipid membranes gives rise to a compressive stress within the bilayers that promotes fluid-to-gel phase transitions. According to the Hydration Forces Explanation, sugars in the intermembrane space minimize the compressive stress and limit increases in the fluid-gel transition temperature, T(m), by acting as osmotic and volumetric spacers that hinder the close approach of membranes. However, the sugars must remain between the bilayers in order to limit the rise in T(m). Large polymers are excluded from the interlamellar space during dehydration and do not limit the dehydration-induced rise in T(m). In this study, we used maltodextrins with a range of molecular weights to investigate the size-exclusion limit for polymers between phosphatidylcholine bilayers. Solutes with sizes ranging from glucose to dextran 1000 limited the rise in lipid T(m) during dehydration, suggesting that they remain between dehydrated bilayers. At the lowest hydrations the solutions vitrified, and T(m) was further depressed to about 20 degrees C below the transition temperature for the lipid in excess water, T(o). The depression of T(m) below T(o) occurs when the interlamellar solution vitrifies between fluid phase bilayers. The larger maltodextrins, dextran 5000 and 12,000, had little effect on the T(m) of the PCs at any hydration, nor did vitrification of these larger polymers affect the lipid phase behaviour. This suggests that the larger maltodextrins are excluded from the interlamellar region during dehydration.     

Probing protein hydration and conformational states in solution.

The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.     

Onion root water transport sensitive to water channel and K+ channel inhibitors

Transroot osmotic water flux (Jos) and radial hydraulic conductivity (Lpr) in onion roots were greatly increased by three means; infiltration of roots by pressurization, repetition of osmosis and chilling at 5 degrees C. Jos was strongly reduced by the water channel inhibitor HgCl2 (91%) and the K+ channel inhibitor nonyltriethylammonium (C9, 75%), which actually made the membrane potential of root cells less sensitive to K+. C9 decreased the rate of turgor reduction induced by sorbitol solution to the same extent as HgCl2. Thus, C9 is assumed to decrease the hydraulic conductivity (Lp) of the plasma membrane by blocking water channels, although possible inhibition of the plasmodesmata of the root symplast by C9 cannot be excluded. Onion roots transported water from the tip to the base in the absence of the osmotic gradient. This non-osmotic water flux (Jnos) was equivalent to Jos induced by 0.029 M sorbitol. Jnos increased when Jos was increased by repetition of osmosis and decreased when Jos was decreased by either HgCl2 or by C9. The correlation between Jnos and Jos suggests that non-osmotic water transport occurs via the same pathways as those for osmotic water transport.     

Binding constants of Li+, K+, and Tl+ in the gramicidin channel determined from water permeability measurements.

In an open circuit there can be no net cation flux through membranes containing only cation-selective channels, because electroneutrality must be maintained. If the channels are so narrow that water and cations cannot pass by each other, then the net water flux through those "single-file" channels that contain a cation is zero. It is therefore possible to determine the cation binding constants from the decrease in the average water permeability per channel as the cation concentration in the solution is increased. Three different methods were used to determine the osmotic water permeability of gramicidin channels in lipid bilayer membranes. The osmotic water permeability coefficient per gramicidin channel in the absence of cations was found to be 6 x 10(-14) cm3/s. As the cation concentration was raised, the water permeability decreased and a binding constant was determined from a quantitative fit to the data. When the data were fitted assuming a maximum of one ion per channel, the dissociation constant was 115 mM for Li+, 69 mM for K+, and 2 mM for Tl+.     

Partial Exclusion between T-Even Bacteriophages; an Incipient Genetic Isolation Mechanism

Conditional lethal mutant systems developed in T-even bacteriophages T2, T4 and T6 have been used to study the partial exclusion which characterizes mixed infections of these phages. In bacteria mixedly infected with T2 and T4, the dominant phage (T4) acts against localized exclusion sensitivity determinants in the genome of the excluded phage (T2). These determinants are clustered near genes controlling early functions; the determinants themselves do not appear among the progeny, but markers located close to them appear infrequently, by recombination. The excluding action of T4 does not depend on the action of any gene so far identified by conditional lethal mutations, nor does it depend on differences in DNA glucosylation between infecting phages. Regardless of mechanism, the genetic consequence of this partial exclusion is to limit genetic exchange between T2 and T4 in the region of the genome controlling early functions, while retaining the capacity for extensive exchange in other regions; in short, partial exclusion constitutes a localized genetic isolating mechanism. Related forms of partial exclusion characterize mixed infections of other T-even phages, including those of some phages newly isolated from nature.     

Probing alamethicin channels with water-soluble polymers. Effect on conductance of channel states.

Channel access resistance has been measured to estimate the characteristic size of a single ion channel. We compare channel conductance in the presence of nonpenetrating water-soluble polymers with that obtained for polymer-free electrolyte solution. The contribution of the access resistance to the total alamethicin channel resistance is approximately 10% for first three open channel levels. The open alamethicin channel radii inferred for these first three levels from the access resistance are 6.3, 10.3, and 11.4 A. The dependence of channel conductance on polymer molecular weight also allows evaluation of the channel dimensions from polymer exclusion. Despite varying conductance, it was shown that steric radii of the alamethicin channel at different conductance levels remain approximately unchanged. These results support a model of the alamethicin channel as an array of closely packed parallel pores of nearly uniform diameter.     

Regulation of glycosaminoglycan function by osmotic potentials. Measurement of water transfer during antithrombin activation by heparin.

The sulfated glycosaminoglycan heparin is an important anticoagulant, widely used to treat and to prevent arterial thrombosis. Heparin triggers conformational changes in, and the functional activation of, the serine proteinase inhibitor antithrombin. We investigated water-transfer reactions during the activation process to explore the possibility that functional interaction between antithrombin and sulfated glycosaminoglycans can be regulated by osmotic potentials. Volume of water transferred upon heparin binding was measured from differences in free energy change, Delta(Delta G), with osmotic stress, pi. Osmotic stress was induced with chemically inert probes that are geometrically excluded from the water-permeable spaces of antithrombin and from intermolecular spaces formed during the association reaction. The free energy change, Delta G, for the antithrombin/heparin interaction was calculated from the dissociation constant, determined by functional titrations of heparin with antithrombin at fixed concentrations of the coagulation protease factor Xa. The effect of osmotic stress was independent of the chemical nature of osmotic probes but correlated with their radius up to radius >17 A. In mixtures including a large and a small probe, the effect of the large probe was not modified by the small probe added at a large molar excess. With an osmotic probe of 4-A radius, the Delta(Delta G)/pi slope corresponds to a transfer of 119 +/- 25 water molecules to bulk solution on formation of the complex. Analytical characterization of water-permeable volumes in x-ray-derived bound and free antithrombin structures revealed complex surfaces with smaller hydration volumes in the bound relative to the free conformation. The residue distribution in, and atomic composition of, the pockets containing atoms from residues implicated in heparin binding were distinct in the bound versus free conformer. The results demonstrate that the heparin/antithrombin interaction is linked to net water transfer and, therefore, can be regulated in biological gels by osmotic potentials.     

Measuring the interaction of urea and protein-stabilizing osmolytes with the nonpolar surface of hydroxypropylcellulose

The interaction of urea and several naturally occurring protein-stabilizing osmolytes, glycerol, sorbitol, glycine betaine, trimethylamine oxide (TMAO), and proline, with condensed arrays of a hydrophobically modified polysaccharide, hydroxypropylcellulose (HPC), has been inferred from the effect of these solutes on the forces acting between HPC polymers. Urea interacts only very weakly. The protein-stabilizing osmolytes are strongly excluded. The observed energies indicate that the exclusion of the protein-stabilizing osmolytes from protein hydrophobic side chains would add significantly to protein stability. The temperature dependence of exclusion indicates a significant contribution of enthalpy to the interaction energy in contrast to expectations from "molecular crowding" theories based on steric repulsion. The dependence of exclusion on the distance between HPC polymers rather indicates that perturbations of water structuring or hydration forces underlie exclusion.     

Effect of polyethylene glycol on the supercoiling free energy of DNA

The supercoiling free energy of pUC19 DNA [2686 base pairs (bp)] was measured in various concentrations of PEG 8000 (polyethylene glycol; molecular weight 8000) by the topoisomer distribution method. The effective twist energy parameter (E(T)) that governs the supercoiling free energy declined linearly by 1.9-fold with increasing w/v % PEG from 0 to 7.5%, which lies below the threshold for intermolecular condensation. In principle, PEG could affect E(T) either via an osmotic exclusion mechanism or by altering the torsion elastic constant, bending rigidity, or self-repulsions of the DNA. Possible alterations of the DNA secondary structure and torsion elastic constant were assessed by CD spectroscopy and time-resolved fluorescence polarization anisotropy of intercalated ethidium. Up to 7.5% PEG, the secondary structure of the DNA remained largely unaltered, as evidenced by (1) the absence of any significant change in the CD spectrum, (2) an extremely small relative decrease (-0.0013) in intrinsic twist, and (3) a negligibly small change in the torsion elastic constant. The observed reduction in E(T) cannot be ascribed primarily to a decrease in torsion elastic constant, and most likely does not stem from a decrease in bending rigidity either. The decrease in medium dielectric constant due to PEG should increase the self-repulsions, and thereby increase E(T), which is opposite to the observed trend. Instead, the observed decline in E(T) is attributed to an osmotic exclusion mechanism. The change in molar volume excluded to the PEG (Delta V(ex)), when the linking difference converts from Delta l = 0 to Delta l = +/-1, was determined from the observed E(T) value and PEG osmotic pressure at each concentration. The experimental Delta V(ex) values agree well with theoretical estimates reckoned for a simple osmotic exclusion model, in which PEG is excluded by hard-core interactions from a concentric cylindrical volume around every duplex segment. The difference in volume excluded to PEG between the Delta l = 0 and the Delta l = +/-1 topoisomers is attributed entirely to the approximately 0.7 additional writhe "crossing" of two duplex strands at roughly 90 degrees, which is known to occur in the latter species. When the separation between the duplex centers at the "crossing" was adjusted so that the theoretical estimate of Delta V(ex) matched the experimental value at each PEG concentration, a value near 5.7 nm was obtained in each case. The invariance and plausible magnitude of this mean separation at the crossing provide strong support for this simple osmotic exclusion model. An alternative model, in which the PEG is excluded from the entire coil envelope of the DNA out to its radius of gyration, perhaps because it decreases the local dielectric constant, was also considered. The estimated difference in excluded volume in that case exceeds the experimental value by a factor of nearly 10(4), and could be ruled out on that basis.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Direct measurement of temperature-dependent solvation forces between DNA double helices.

The assembly of double stranded DNA helices with divalent manganese ion is favored by increasing temperature. Direct force measurements, obtained from the osmotic stress technique coupled with x-ray diffraction, show that the force characteristics of spontaneously precipitated Mn(2+)-DNA closely resemble those observed previously by us for other counterion condensed DNA assemblies. At temperatures below the critical one for spontaneous assembly, we have quantitated the changes in entropy and manganese ion binding associated with the transition from repulsive to attractive interactions between helices mediated by osmotic stress. The release of structured water surrounding the DNA helix to the bulk solution is the most probable source of increased entropy after assembly. Increasing the water entropy of the bulk solution by changing the manganese salt anion from CI- to ClO4- predictably and quantitatively increases the transition entropy. This is further evidence for the dominating role of water in the close interaction of polar surfaces.     

Application of a thermodynamic model to the prediction of phase separations in freeze-concentrated formulations for protein lyophilization

Many of the compounds considered for use in pharmaceutical formulations demonstrate incompatibilities with other components at high enough concentrations, including pairs of polymers, polymers and salts, or even proteins in combination with polymers, salts, or other proteins. Freeze concentration can force solutions into a region where incompatibilities between solutes will manifest as the formation of multiple phases. Such phase separation complicates questions of the stability of the formulation as well as labile components, such as proteins. Yet, phase separation events are difficult to identify by common formulation screening methods. In this report, we use the osmotic virial expansion model of Edmond and Ogston (1) to describe phase-separating behavior of ternary aqueous polymer solutions. Second osmotic virial coefficients of polyethylene glycol 3350 (PEG) and dextran T500 were measured by light scattering. Assuming an equilibrium between ice and water in the freeze-concentrated solution, a degree of freeze concentration can be estimated, which, when combined with the phase separation spinodal, describes a "phase separation envelope" in which phase separation tendencies can be expected in the frozen solution. The phase separation envelope is bounded at low temperatures by the glass transition temperature of the freeze-concentrated solution. Scanning electron microscopic images and infrared spectroscopy of protein structure are provided as experimental evidence of the phase separation envelope in a freeze-dried system of PEG, dextran, and hemoglobin.     

Water and nonelectrolyte permeability of plant cell membranes after short term application of amino acids and phosphorylated amino acids

The effects of amino acids (aa) and N-(diisopropyloxyphosphoryl)-amino acids (DIPP-aa) on cell membranes were investigated by evaluating water and methyl urea permeability. Permeability coefficients P_f and P_s were determined by standard osmotic methods for cells ofPisum sativum stem base epidermis after 20 min exposure to a 5 mM solution of each aa and DIPP-aa. The P_f value ofP. sativum epidermal cells (untreated controls) was 1.3 ± 0.4 × 10^-3 μm s^-1. Treat ments with the diisopropyl-oxyphosphoryl derivatives of three one charged and three polar amino acids (serine, threonine, asparagine, and aspartic acid) and unsubstituted (free) serine and threonine increased water permeability up to about two fold of the control value. Serine and threonine and their DIPP-derivatives increased methyl urea permeability (controls 1.03 ± 0.09 × 10^-3 μm s^-1) 30 to 80 percent Other amino acids and their DIPP-derivatives caused small or insignificant changes of water permeability. Only certain polar amino acids and their DIPP-derivatives increased the osmotic water and methyl urea permeation through the plasma membrane. The specificity of these molecules on plasma membranes suggests that the active amino acids (serine and threonine) and their DIPP-derivatives interact with charged membrane molecules. The relatively small changes in water and methyl urea permeability may indicate that the effective aa’s and their DIPP-derivatives interact with phospholipids rather than aquaporin. A concurring alteration of water channel proteins, however, cannot excluded.     

Exclusion probabilities for pedigree testing farm animals

Pedigree testing, using genetic markers, may be undertaken for a variety of situations, of which the classical paternity testing is only one. This has not always been made clear in the literature. Exclusion probabilities associated with various testing situations, including the use of autosomal or X-linked codominant marker systems with any number of alleles, are presented. These formulae can be used to determine the appropriate exclusion probability for the situation being investigated. One such situation is where sire groups of progeny are to be verified without knowledge of the dams' genotypes, in which case the classical paternity exclusion probability is too high, and if used may result in an optimistic declaration about the progeny that have not been excluded. On the other hand, if mating pairs are known then incorrect progeny can be excluded at a higher rate than suggested by paternity exclusion calculations. The formulae also assist in determining the usefulness of X-linked markers, particularly if the pedigree checks involve progeny of only one sex. A system of notation that is useful for the algebraic manipulation of genetic probabilities, including exclusion probabilities as presented here, is also given.     

Extent and properties of nonbulk "bound" water in crystalline lens cells

Crystalline lenses provided good material to study and measure the properties of cellular water. Different methods were used to establish the extent and properties of nonbulk water in mammalian lenses. These methods include: NMR titration analysis, a test of the osmotic properties, a test of dye exclusion In lenses with intact cell membranes and in lenses with disrupted cell membranes, and the water-holding capacity of lenses subjected to 40,000 x g for 1 hour with intact cell membranes and in lenses with disrupted cell membranes. The data from these methods, as well as other data from the literature, lead to the conclusion that most, if not all, of the water in lens cells (up to 2.2 g water/g dry mass) has motional and osmotic properties that distinguish it from bulk water. These findings call into question the common and convenient assumption that all but a small proportion of cellular water is like that in dilute solution.     

Contribution of solvent water to the solution X-ray scattering profile of proteins

A theoretical framework is presented to analyze how solvent water contributes to the X-ray scattering profile of protein solution. Molecular dynamics simulations were carried out on pure water and an aqueous solution of myoglobin to determine the spatial distribution of water molecules in each of them. Their solution X-ray scattering (SXS) profiles were numerically evaluated with obtained atomic-coordinate data. It is shown that two kinds of contributions from solvent water must be considered to predict the SXS profile of a solution accurately. One is the excluded solvent scattering originating in exclusion of water molecules from the space occupied by solutes. The other is the hydration effect resulting from formation of a specific distribution of water around solutes. Explicit consideration of only two molecular layers of water is practically enough to incorporate the hydration effect. Care should be given to using an approximation in which an averaged electron density distribution is assumed for the structure factor because it may predict profiles considerably deviating from the correct profile at large K.