The effect of pH on colchicine conformation and structure.

The effect of various pH values between 0 and 14 on the structure and conformation of colchicine was examined using UV-vis spectrophotometry at a concentration of 1.7 x 10(-5) M and NMR techniques at a colchicine concentration of 0.1M. The complete interpretation of the colchicine NMR spectrum in D2O is given. A stable structure of the colchicine molecule in aqueous solutions at pH from 2 to 12 was demonstrated. However, during incubation at 40 degrees C colchicine was found to be stable only at pH values between 2 and 10. The significance of these data for reactions of cholchicine in regard to metabolism and interaction with macromolecules is discussed.     

Effect of pH on the conformation and stability of the plant toxin ricin

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.     

Plastocyanin conformation. The effect of nitrotyrosine modification and pH

Plastocyanin isolated from several species including spinach, poplar, and lettuce showed conformational changes both upon reduction and upon lowering the pH as determined by near-ultraviolet absorption and fluorescence measurements. The fluorescence excitation maximum was at 278 nm for all species of plastocyanin measured. In the case of spinach, the emission maximum was at 310–312 nm, similar to a tyrosine residue in solution. The fluorescence intensity increased 22% upon reduction of plastocyanin at pH 7.0. In poplar plastocyanin, the emission maximum was shifted to 335 nm and increased only 10% upon reduction. The 335 nm emission peak observed in poplar plastocyanin is attributed to Tyr 80 which is hydrogen bonded to a carbonyl group on the protein backbone. Tyr 83 was also shown to undergo fluorescence changes upon reduction since the redox state-dependent fluorescence changes decreased for a nitrotyrosine (nitrotyrosine-plastocyanin) derivative of this residue. These results show that the east face of the molecule, which contains both Tyr 80 and 83 as well as a possible binding site [1,2], undergoes conformational changes upon reduction. These conformational changes may be involved in promoting smooth electron transport between plastocyanin and its reaction partners. Both the absorption and fluorescence were found to be pH dependent. The quantum yield for fluorescence increased sharply below pH 6 for both oxidized and reduced spinach plastocyanin. This may be related to the appearance of a redox-inactive form of reduced plastocyanin [3]. The conformational changes observed at low pH may provide a mechanism for control of electron transport by the proton gradient. Low concentrations of CaCl₂ (10 mM) had no effect on plastocyanin fluorescence. However, addition of 2.7 M (NH₄)₂SO₄ eliminated the redox-dependent fluorescence changes.     

Influence of pH on the conformation and stability of mismatch base-pairs in DNA

A series of self-complementary dodecanucleotide duplexes containing two symmetrically disposed mismatches have been studied by pH-dependent, ultraviolet light melting techniques. The results indicate that A.C, and C.C mismatches are strongly stabilized by protonation and that the degree of stabilization of the A.C mismatch depends greatly on the flanking bases. In one case, a duplex containing two A.C mismatches is more stable than the native sequence below pH 5.5. The G.A mismatch displays conformational flexibility, with a protonated G(syn).A(anti) base-pair occurring in certain base stacking environments but not in others. The A.A and T.C mismatches are not stabilized at low pH. These solution studies correlate well with predictions based on X-ray crystallographic data.     

The effect of plant succession on slope stability

The aim of this field investigation was to study the enrichment of biodiversity of the slope at an early phase of succession, initiated by selected pioneers, and to study how this enrichment related to enhancement of the slope stability. Four experimental plots, with differing plant pioneers and number of species (diversity), were designed in order to assess the effects of plant succession on slope stability. Plant growth pattern was assessed by observing the increment in species diversity (number), species frequency and plant biomass. Higher vegetation biomass in a mixed culture situation (LLSS) in the field with Leucaena leucocephala as a pioneer, marked an increase in species diversity after 24 months of observation. In contrast, G (grasses and legume creepers) plot revealed the slowest rate of succession and the lowest above-ground biomass amongst the plots. The mixed-culture plot without L. leucocephala (SS) had also shown a lower biomass, a similar phenomenon observed in a plot grown by L. leucocephala (LL) with low plant diversity. Consequently, these plant growth patterns gave a positive effect on slope stability where the regression study showed that the shear strength was much affected by plant biomass. Meanwhile, throughout the succession process in LLSS plot, root length density reached the highest value amongst the plots, 23 Km m^?3. In relation to this, the saturation level of the slope indicates the unsaturated condition of the soil which resulted in the enhancement of both soil penetrability and soil shear strength of the plot. These attributes reveal a strong positive relationship between the process of natural succession and the stability of slopes.     

Estimation of pH effect on the structure and stability of kinase domain of human integrin-linked kinase

Integrin-linked kinase (ILK) is an evolutionarily conserved Ser/Thr protein kinase, involved in many physiological functions such as signal transduction, actin rearrangement, cell proliferation, migration, polarisation, angiogenesis and apoptosis. An increased expression of ILK is associated with different cancers and thus considered as an attractive target for cancer therapy. We have successfully cloned, expressed and purified the kinase domain (193–446 residues) of ILK. To see the effect of pH on the structure and conformation, we performed circular diachroism, fluorescence and absorbance measurements in a wide range of pH conditions. We observed that within the range of pH 7.5–11.0, ILK_193–446 maintains its both secondary and tertiary structures. While visible aggregates were observed under the acidic pH 2.0–5.5 conditions, in order to complement these observations, we have performed molecular dynamics simulations of this kinase domain by mimicking diverse pH conditions which enabled us to see conformational preferences of the protein under such conditions. A significant correlation between the spectroscopic and molecular dynamics simulation was observed. These findings are useful to understand the conformation of ILK protein under certain pH condition which may be further implicated in the drug design and discovery.     

The effect of structure and conformation on the relative hydrophobicity of dinucleosidephosphates and their analogs

Partition of a number of dinucleosidephosphates, their (2'-5')-isomers and a series of their analogues, in which the ribose ring was replaced by acyclic hydroxyalkyl substituents, in an aqueous biphasic ficoll--dextran system has been studied. Effect of the ionic composition of the biphasic system on partitioning of the compounds was examined. The relative hydrophobicity of the compounds in the presence of 0.11 M phosphate buffer, pH 7.4, and in the presence of 0.15 M NaCl in 0.01 M buffer, pH 7.4, was estimated. The results obtained are considered in regard to the effect of structure and conformation of a molecule on its affinity for an aqueous environment.     

Numerical calculations of the pH of maximal protein stability. The effect of the sequence composition and three-dimensional structure.

A large number of proteins, found experimentally to have different optimum pH of maximal stability, were studied to reveal the basic principles of their preference for a particular pH. The pH-dependent free energy of folding was modeled numerically as a function of pH as well as the net charge of the protein. The optimum pH was determined in the numerical calculations as the pH of the minimum free energy of folding. The experimental data for the pH of maximal stability (experimental optimum pH) was reproducible (rmsd = 0.73). It was shown that the optimum pH results from two factors - amino acid composition and the organization of the titratable groups with the 3D structure. It was demonstrated that the optimum pH and isoelectric point could be quite different. In many cases, the optimum pH was found at a pH corresponding to a large net charge of the protein. At the same time, there was a tendency for proteins having acidic optimum pHs to have a base/acid ratio smaller than one and vice versa. The correlation between the optimum pH and base/acid ratio is significant if only buried groups are taken into account. It was shown that a protein that provides a favorable electrostatic environment for acids and disfavors the bases tends to have high optimum pH and vice versa.     

The effect of carbohydrate on the structure and stability of erythropoietin.

Erythropoietin is a glycoprotein hormone that stimulates the maturation of late erythroid progenitor cells. It has three N-linked and one O-linked carbohydrates which play an important role in the biosynthesis and biological activities of the protein. To determine the role the carbohydrate might have in maintaining the conformational stability of the protein, the protein expressed in mammalian cells (fully glycosylated), the asialo mammalian-expressed protein, and the protein expressed in Escherichia coli (no carbohydrate) were compared for their stability to guanidine HCl, pH, and temperature. Circular dichroism was used to follow protein unfolding. Both the intact and asialo mammalian-expressed proteins unfolded with a cooperative transition in guanidine HCl, with a midpoint at 1.75 M guanidine HCl. The E. coli-expressed material unfolded with a midpoint of 1.2 M guanidine HCl, and a delta G of unfolding which was 1.4 kcal/mol less than that of the two glycosylated molecules. The E. coli-derived protein was also significantly less stable to pH-induced conformational changes, showing a cooperative transition in 35% glycerol with a midpoint at pH 4.4, while both the intact and asialo mammalian-expressed molecules had a transition midpoint of pH 3.75 in the absence of glycerol, and approximately pH 3 in the presence of 35% glycerol. The E. coli-expressed molecule unfolded and precipitated upon heating to 44 degrees C, while the asialo and intact mammalian-expressed proteins remained soluble, with a Tm of 56 degrees C. From these experiments, the carbohydrate appears to play a critical role in stabilizing the erythropoietin molecule to denaturing conditions, and this increased stability does not depend on the presence of sialic acid.     

The effect of environmental stability on hyporheic community structure

Interstitial water samples were collected from two well depths (30 and 60 cm) from 8 rivers in the Cass-Craigieburn region, South Island (New Zealand) during January 1998 to assess the hyporheos in streams of differing stability. Hyporheic water chemistry was more similar to surface water chemistry at unstable sites than at stable sites. The greatest diversity of both epigean and hypogean animals occurred at intermediate levels of disturbance; however, invertebrate density declined with increasing bed movement. Abundance of epigean animals also increased with declining environmental stability (i.e., increasing bed movement). Water chemistry was most like the river channel at unstable sites, probably because less stable substrates had increased interstitial flow facilitating a greater supply of water from the surface channel. In contrast, stable sites were spring fed and groundwater had a greater influence. Thus, substrate stability may influence hyporheic water chemistry and porosity by changing the interaction between ground and surface waters. Epigean taxa may be less abundant at stable sites because the chemical nature of the water is least like the river channel (e.g. low dissolved oxygen, higher temperature and higher conductivity), while the abundance of hypogean animals is greater, as these taxa are more tolerant of this water chemistry, and are thus capable of persisting in the stable substrates.     

The effect of solvent environment on the conformation and stability of human polyclonal IgG in solution.

Stability of therapeutic IgG preparations is an important issue as adequate efficacy and safety has to be ensured throughout a long shelf life. To this end, denaturation and aggregation have to be avoided. In many cases sugars are applied for stabilizing IgG in relatively high concentration (5-10%). However, certain sugars (sucrose, maltose) are responsible for adverse effects including renal failure. In this work we reassessed the effect of pH and stabilizers to optimize the solvent environment and minimize the amount of additives without endangering quality and stability. Since both biological function and aggregation depend on the conformational properties of individual IgG molecules, two sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of pH and addition of standard stabilizers. It was observed that the conformational stability decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage is 5.0-6.0, as a compromise between conformational stability and the tendency for oligomerization. Intriguingly, additives in physiologically acceptable concentration have no effect on the thermal stability of IgG. On the other hand, glucose or sorbitol, even at a concentration as low as 1%, have significant effect on the tertiary structure as revealed by near-UV-CD spectroscopy, reflecting changes in the environment of aromatic side-chains. Although, 0.3% leucine does not increase conformational stability, it decreases the aggregation tendency even more efficiently than 1% glucose or sorbitol. Both pH and storage temperature are decisive factors for the long-term stability of IgG solutions. An increase in the dimer content was observed upon storage at 5 degrees C which was partly reverted upon incubation at 37 degrees C. Storage at temperatures higher than 5 degrees C may help to maintain an optimal proportion of dimers. Regarding the known side effects, and their limited stabilizing capacity at low concentration, it is advisable to omit sugars at intravenous immunoglobulin (IVIG) formulation. Hydrophobic amino acids give promising alternatives.     

Electromechanical stresses and the effect of pH on membranes structure

The presence of an electric field in a membrane creates stresses which lead to a mechanical compression of the membrane material.It is shown that considerations of this effect yield results consistent with the observed differential effect of pH on the plasma membrane substructure if the central electron-lucent layer in OsO₄-fixed membranes is identified with the ionic depletion in the double-fixed charge model of cell membranes.     

The effect of iodide and chloride on transthyretin structure and stability

Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.     

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides.

The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1. Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2, which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1, especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2. This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.     

The effect of Ptilotus plant tissue on pH of in vitro media

Changes occur in the pH of in vitro nutrient media during preparation and over the culture period. The direction and extent of the changes depend upon the initial pH and the presence or type of gelling agent. Agar-based medium was progressively acidified in the presence of a living Ptilotus exaltatus explant. This was not a response to wounding and the rate of acidification could be maintained by frequent replacement of the medium. The rate appears to be pH dependent with an equilibrium occurring at pH4. The pH effect was not accounted for by K⁺ uptake.     

The effect of pH on biological activity of plant cytotoxin cauloside C

The effect of plant carboxyl-containing glycoside cauloside C upon eucaryotic cells has been studied. The glycoside interacts with cells as a pH-dependent cytotoxin and increases K+ leakage and Ca2+ uptake with strong action in acidic media Cell viability after glycoside action at acidic pH may be recovered by the shift of medium pH from 5.6 to 7.4. Directed transport of low molecular weight effectors such as cAMP and Ca2+ to human embryo fibroblasts under the action of cauloside C has been demonstrated. Calcium uptake is accompanied by about a twofold stimulation of fibroblast proliferation in serum-free medium. The manifestation of the effect depends on the strictly determined time of the 'open' state of the membrane permeability (2 min) and upon concentration of glycoside in the medium (1 ng/ml) Cauloside C-stimulated Ca-transport is not blocked by Ca-channel blockers such as verapamil, diltiasem, and nitrendipine (all at a concentration of 1 x 10(-6) M) but these blockers inhibit cauloside C-stimulated proliferation of fibroblasts. We conclude that stimulation of fibroblast proliferation is caused by activation of membrane associated Ca-channels at the expense of calcium, incorporated into cells with cauloside C. The use of cauloside C as a new biochemical tool for cell permeabilisation is suggested.     

The effect of pH on the structure and activity of yeast RNA polymerase I

The analysis of the effect of pH upon the rate of polymerization indicates that the activity of yeast RNA polymerase I is optimal between pH 7.5 and 9 and depends on the ionization state of two groups with apparent pK_a values of 6.5 and 10. Yeast RNA polymerase I is extremely labile at acid pH. Below pH 5 the enzyme is irreversibly inactivated by [H⁺], with a second-order rate constant of 1.6 × 10^?4m^?1 min^?1. Sucrose gradient sedimentation and gel electrophoresis analysis of the enzyme inactivated at acid pH indicates the sequential dissociation of several enzyme subunits. The polypeptides of 44,000 and 24,000 daltons dissociate first from the enzyme core followed by the dissociation of the polypeptides of 48,000 and 36,000 daltons.     

The conformation properties and conformation stability of streptokinase and its polymer derivative

The properties and conformational stability of the proteinaceous activator of fibrinolysis--native streptokinase--and its derivative obtained by modification with a linear hydrophilic copolymer based on N-vinylpyrrolidone, were studied by the circular dichroism method. It was shown that polymeric modification of streptokinase had no effect on the secondary structure, while the conformational stability of the modified protein to urea was higher than that of the native one. Studies on thermal stability of both native and modified forms of streptokinase showed that the inactivation rate was lower in the modified form as compared to the native one.