Cryoenzymologic studies on arginine kinase: solvent, temperature and pH effects on the overall reaction.

The overall reaction catalyzed by the phosphotransferase arginine kinase was studied at normal and subzero temperatures. Ethylene glycol was used as the antifreeze and its effects on the Km values of substances, kcat and pH profiles were investigated in detail. a) The Km values for the substrate (2 mM for ATP and 0.6 mM for arginine) were little affected by the solvent composition or temperature of the reaction mixture. b) At concentration of ethylene glycol higher than 40% there was a sharp drop of enzyme activity. c) Ethylene glycol induces a large shift in the enzymic pK D) At -5 degrees C in 40% of solvent there was a break in the Arrhenius plot suggesting a change of the rate-limiting step. The relevance of these results to the reaction pathway of arginine kinase is discussed. In addition, controlled perturbations induced by cosolvent and temperature appear as useful tools for further kinetic investigations.     

Stability of proteins: temperature, pressure and the role of the solvent

We focus on the various aspects of the physics related to the stability of proteins. We review the pure thermodynamic aspects of the response of a protein to pressure and temperature variations and discuss the respective stability phase diagram. We relate the experimentally observed shape of this diagram to the low degree of correlation between the fluctuations of enthalpy and volume changes associated with the folding-denaturing transition and draw attention to the fact that one order parameter is not enough to characterize the transition. We discuss in detail microscopic aspects of the various contributions to the free energy gap of proteins and put emphasis on how a cosolvent may either enlarge or diminish this gap. We review briefly the various experimental approaches to measure changes in protein stability induced by cosolvents, denaturants, but also by pressure and temperature. Finally, we discuss in detail our own molecular dynamics simulations on cytochrome c and show what happens under high pressure, how glycerol influences structure and volume fluctuations, and how all this compares with experiments.     

Studies on the properties of immobilized urokinase: effects of pH and temperature

Human urokinase was immobilized on an ethylene vinyl acetate copolymer surface. Soluble urokinase showed its maximum activity at pH 8.5, while the immobilized enzyme was most active at pH 9.0. Apparently, the shift in optimal pH was due to the polyanionic nature of the carrier surface on which the enzyme was immobilized. Optimal temperatures of soluble urokinase and immobilized enzyme were identical, i.e., 37 degrees C. The stability of immobilized enzyme against thermal degradation was several times higher than that of the soluble enzyme. Its stability at higher temperatures is one of the main reasons for the clinical use of immobilized urokinase as an antithrombotic material.     

Nucleosome conformation: pH and organic solvent effects.

Monomer nucleosomes (nu 1) from chicken erythrocyte nuclei were examined in aqueous buffers (8 greater than pH greater than 3) and in solvent mixtures (i.e., water and ethanol, ethylene glycol, dioxane, dimethyl sulfoxide, 2-methyl-2,4-pentanediol, polyethylene glycol, sucrose, or urea). Circular dichroism, laser Raman spectroscopy of nu 1, and the fluorescence of nu 1 labeled with N-(3-pyrene) maleimide on thiol groups of H3 histone were employed to detect conformational transitions in nu 1. The results of pH studies were as follows: 5.5 greater than pH greater than 4.8, suppression of DNA ellipticity and no change of histone alpha-helix; 4.6 greater than pH greater than 4.2 an irreversible increase in the B character of DNA, a slight loss of histone alpha-helix, and a parallel loss of pyrene excimer fluorescence; 4 greater than pH, aggregation of nu 1 and protonation of the DNA bases C and A. Results obtained in the studies of nu 1 in solvent mixtures included the following: sharp conformational transitions that variously involved an increase in the B character of DNA, a slight loss of histone alpha-helix, and a loss of pyrene excimer. Different solvents required different concentrations to effect these conformational changes.     

Random coil chemical shift for intrinsically disordered proteins: effects of temperature and pH

Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins. The quality of the secondary chemical shifts is dependent on an appropriate choice of random coil chemical shifts. We report random coil chemical shifts and sequence correction factors determined for a GGXGG peptide series following the approach of Schwarzinger et al. (J Am Chem Soc 123(13):2970–2978, 2001). The chemical shifts are determined at neutral pH in order to match the conditions of most studies of intrinsically disordered proteins. Temperature has a non-negligible effect on the ¹³C random coil chemical shifts, so temperature coefficients are reported for the random coil chemical shifts to allow extrapolation to other temperatures. The pH dependence of the histidine random coil chemical shifts is investigated in a titration series, which allows the accurate random coil chemical shifts to be obtained at any pH. By correcting the random coil chemical shifts for the effects of temperature and pH, systematic biases of the secondary chemical shifts are minimized, which will improve the reliability of detection of transient secondary structure in disordered proteins.     

The effects of pH and temperature on the kinetic properties of skeletal muscle lactate dehydrogenase from anuran amphibians

Summary Muscle LDH activities were measured in two anuran amphibians with different behaviour and ecology, Rana perezi and Bufo calamita. Both pyruvate reduction and lactate oxidation were measured at temperatures of 15, 20 and 30°C, and at pH 7.0, 7.4, and 8.0. Pyruvate and lactate muscle concentrations were determined in individuals at rest and after exercise. R. perezi muscle used anaerobic glycolysis during 3 min of exhaustive exercise, with rising pyruvate and lactate concentrations. Enforced walking for 30 min caused high variability in lactate concentration in B. calamita muscle. Temperature and pH changes affected apparent Km values for pyruvate. When these factors varied simultaneously, enzyme affinity tended not to change. Thus, the thermodynamic effect on pyruvate reduction activity is high, especially at physiological substrate concentrations. In contrast, lactate oxidation activity tended to stabilize when temperature and pH varied jointly. Inhibition by substrate, pyruvate or lactate, seemed to have no importance in vivo.During exercise there was a rise in pyruvate concentration, and a probable decrease in pH, which increased pyruvate reduction reaction and decreased lactate oxidation, contributing to lactate accumulation in Rana perezi muscle. B. calamita muscle did not show pyruvate increase after exercise and its LDH was less dependent on pH at physiological concentrations. Pyruvate reduction rate did not therefore increase. R. perezi muscle enzyme had features of anaerobic LDH while B. calamita LDH muscle was more similar to mammalian heart enzyme, with differences in accordance with the different behaviour of these anurans.Abbreviations LDH lactate dehydrogenase     

Steady-state properties of calcium binding to parvalbumins from bullfrog skeletal muscle: effects of Mg2+, pH, ionic strength, and temperature

To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI 4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I = 0.106, pH 6.80, at 20 degrees C) containing various concentrations of Mg2+ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca2+ in the presence of Mg2+ changed in the manner expected if Ca2+ and Mg2+ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, KCa = 1 X 10(7) M-1, KMg = 900 M-1; for PA-2, KCa = 6 X 10(6) M-1, KMg = 830 M-1 (I = 0.106, pH 6.80, at 20 degrees C). The apparent binding constants are strongly dependent on temperature: at 10 degrees C for PA-1, KCa = 2 X 10(8) M-1, KMg = 10(4) M-1; for PA-2, KCa = 5 X 10(7) M-1, KMg = 5 X 10(3) M-1 (I = 0.106, pH 6.80). The dependence of the affinities for Ca2+ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca2+ and Mg2+ of parvalbumins are unchanged between pH 6.5 and 7.2.     

Specificity of acyl-CoA:phospholipid acyltransferases: solvent and temperature effects

Acyltransfer from CoA thiol esters to either the 1- or 2-position of monoacylglycerophosphoryl choline, which is catalyzed by a microsomal preparation from rat liver, had a temperature optimum of 30-35 degrees C. No significant alteration was observed in the ability of the acyltransferases to distinguish among the various thiol esters tested in the range of 15-40 degrees C. Acyl-CoA:1-acylglycerophosphoryl choline acyltransferase activity is inhibited by urea, N-alkyl ureas, and short-chain alcohols. The effect is not equal for all acyl derivatives, and ethylene glycol has much less inhibitory effect on the transfer of acids with an n - 6 (omega6) double bond. On the other hand, this inhibition of acyltransfer was relatively insensitive to the configuration of the Delta(9)-double bond of octadecadienoates. The specificity of the enzyme-catalyzed transfer of different acids to the 2-position can be correlated in part with the dissociation constants for the urea clathrate complexes. Added glycol does not appreciably alter the specificity of enzyme-catalyzed transfer to the 1-position, but it inhibits the transfer of all acids in a similar fashion.     

The internal equilibrium of the hairpin ribozyme: temperature, ion and pH effects

The hairpin ribozyme reversibly cleaves phosphodiesters of RNA substrates to generate products with 5' hydroxyl and 2',3'-cyclic phosphate termini. We previously found that the rate constant for ligation is tenfold faster than the rate constant for cleavage under standard conditions. The hammerhead ribozyme catalyzes the same reactions but is reported to favor cleavage relative to ligation by more than 100-fold under the same conditions. To explore the basis for this difference, we examined the influence of temperature, ions and pH on the hairpin ribozyme internal equilibrium. Under the same conditions, the loss of entropy associated with ligation is less for the hairpin than for the hammerhead ribozyme, consistent with the notion that a more rigid hairpin structure undergoes a smaller decrease in dynamics upon ligation than the more flexible hammerhead structure. Increased salt and reduced temperature shift the equilibrium toward ligation while pH has little effect, suggesting that conditions that stabilize RNA structure tend to promote ligation. The hairpin ribozyme appears to take up at least one tri- or divalent cation or two monovalent cations upon ligation. The efficiency with which different cations promote ligation depends strongly on valence and, less strongly, on ionic radius or electronegativity. This pattern of cation selectivity suggests that cations promote ligation through delocalized electrostatic shielding, perhaps interacting with a region of especially high charge density in the ligated ribozyme. Changes in ionic conditions produce large but compensating changes in enthalpy and entropy for cleavage and ligation. Thus, in addition to any increase in ribozyme dynamics associated with cleavage, re-organization of associated cations contributes significantly to hairpin ribozyme thermodynamics.     

Cadmium and zinc removal from aqueous solutions by Bacillus jeotgali: pH, salinity and temperature effects

Effects of pH, salinity and temperature on biosorption of Cd and Zn by bacteria Bacillus jeotgali strain U3 were evaluated in batch experiments. Traditional and Subsequent Addition Methods (SAM) were used to carry out the bioassays. Sorption of metals was higher when pH or temperature was increased, or when salinity was reduced. The Langmuir isotherm better fit the biosorption data for Cd, while the Freundlich model fitted better for Zn biosorption. A comparison with similar biosorbents suggested that Bacillus jeotgali strain U3 could be considered a good biosorbent for Cd and Zn recovery.     

Oxygenation properties of hemoglobin from the earthworm, Lumbricus terrestris. Effects of pH, salts, and temperature

Oxygen equilibrium curves of the extracellular hemoglobin from Lumbricus terrestris were determined under a variety of conditions. These data were characterized by (i) a rather small free energy of cooperativity (1.6-2.8 kcal/mol), (ii) a large and strongly pH-dependent Hill coefficient with a maximum value of 7.9, (iii) a high sensitivity of the upper asymptote of the Hill plot to pH, and (iv) a maximum association constant as large as that of the free beta subunit of human hemoglobin A. The effects of LiCl, KCl, NaCl, BaCl2, CaCl2, SrCl2, and MgCl2 on the oxygen equilibrium were measured. Cations, not Cl-, were found to control oxygen binding. Divalent cations have a larger effect on oxygen affinity than monovalent cations, and their effectiveness decreased in the order listed above within each valence class. These specific effects depend in part on ionic radius and cannot be explained in terms of ionic strength. The data indicate that the oxygenation-linked binding of a Ca2+ ion is accompanied by the release of two protons; the binding of a Na+ ion is associated with the release of one proton. These findings indicate that the oxygenation-linked cation-binding site contains two acid groups that do not readily dissociate their protons except when replaced by cations. Incubation at either pH 6.2 or 8.9 had no effect on subsequent measurements of oxygen equilibria at pH 7.8. The apparent heat of oxygenation was found to be -11.8, -7.3, and -9.3 kcal/mol at pH 9.0, 7.4, and 6.6, respectively. These differences indicate that proton-binding processes contribute to the heat of oxygenation.     

Chiral recognition ability of curdlan triacetate: solvent and temperature effects

The chiral recognition ability of the chiral stationary phase (CSP) consisting of curdlan (beta-1,3-glucan) triacetate coated on silica gel was clearly changed by the contacting solvents and heat treatment. The chiral recognition ability significantly decreased, particularly at temperatures above 45 degrees C, depending on the racemates. The molecular weight of the curdlan triacetate slightly influenced its ability. The recognition abilities of curdlan tricetate that was lost by heat treatment were partially recovered by contact with methanol. However, when it was contacted with ethanol a different selectivity was observed. The labile chiral recognition ability of curdlan triacetate is in striking contrast to the very stable chiral recognition of cellulose (beta-1,4-glucan) triacetate (Chiralcel OA). This difference may be ascribed to the conformational stability of the acetates consisting of curdlan (beta-1,3-glucan) and cellulose (beta-1,4-glucan) with different sugar linkages.     

Intramolecular cleavage of LexA and phage lambda repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent

LexA repressor of Escherichia coli and phage lambda repressor are inactivated in vivo and in vitro by specific cleavage of an Ala-Gly peptide bond in reactions requiring RecA protein. At mildly alkaline pH, the in vitro cleavage reaction also proceeds spontaneously, suggesting that peptide bond hydrolysis is an activity of the repressors rather than of RecA. The spontaneous cleavage reaction, termed "autodigestion", has been characterized for the LexA and lambda repressors. The results show that the reaction is intramolecular. The rate of LexA autodigestion was studied over the pH range 7.15-11.77 and over the temperature range 4-46 degrees C. The logarithm of the rate constant increased linearly with pH and reached a plateau value (2.5 X 10(-3) s-1 at 37 degrees C) at pH above 10. The data closely followed a model in which a single residue side chain (apparent pK = 9.8 at 37 degrees C) must be deprotonated for the protein to show activity. Analysis of the temperature dependence gave the heat of proton dissociation as 19.9 kcal/mol and the heat of activation for hydrolysis as 15.3 kcal/mol at 25 degrees C. Autodigestion of lambda repressor, studied over the pH range 8.65-10.70 at 37 degrees C, was similar to the LexA reaction in its pH dependence, yielding a pK of 9.8. The maximum rate at 37 degrees C for lambda repressor, 6.1 X 10(-5) s-1, was 40 times slower than for LexA, a difference similar to that previously observed in vivo and in vitro for RecA-dependent cleavage reactions. There was no significant solvent deuterium isotope effect on the autodigestion of LexA. Changes in buffer composition, including high concentrations of glycine for lambda repressor and of imidazole or hydroxylamine for LexA, indicated that solvent components other than water do not participate in the rate-determining step. Removal or addition of metal ions did not significantly affect LexA autodigestion. These and other observations suggest that the deprotonated form of an amino acid side chain plays a central role in the chemistry of the cleavage reaction. The above observations establish repressor autodigestion as a member of an emerging set of biologically important self-processing reactions.     

Peptoid microsphere coatings: The effects of helicity,temperature, pH,and ionic strength

Peptoids are peptidomimetic oligomers that predominantly harness similarities to peptides for biomimetic functionality. They have potential for use in biomedical applications and biosensors due to resistance to proteolytic degradation and low immunogenicity. The incorporation of chiral, aromatic side chains in the peptoid sequence allows for the formation of distinct secondary structures and self-assembly into supramolecular assemblies, including microspheres. Peptoid microspheres can be coated onto substrates for potential use in biosensor technologies, tissue engineering platforms, and drug-delivery systems. In order to be useful for these applications, the peptoid coatings must be robust under physiological conditions. In this study, we report the effects of various conditions on the peptoid microsphere coatings, including (i) helicity, (ii) temperature, (iii) pH, and (iv) ionic strength. These studies show that microsphere size decreases with increasing peptoid helicity and the positively charged side chains are positioned on the outside of the microspheres. The peptoid microsphere coatings are robust under physiological conditions but degrade in acidic conditions (pH < 7) and at low ionic strengths (<150 μM).     

Circular dichroism of nerve membrane fractions: effects of temperature, pH and electrolytes

Abstract— We have demonstrated significant differences in the circular dichroic (CD) spectra of myelin, synaptic vesicle and synaptosomal membrane preparations from rat brain. Although the measured CD spectra probably contained components attributable to turbidity, we have presented arguments to indicate that turbidity effects could not account for all the observed differences. We think it likely, therefore, that these three membrane fractions exhibit intrinsically different CD spectra, and therefore contain proteins of differing three-dimensional structure. The perturbation of membrane CD produced by electrolytes appeared to involve specific ionic effects. Here again, effects of turbidity were probably involved. The increase in light scattering was demonstrated by an increase in A₂₅₀ at increasing salt concentrations. The increase in A₂₅₀ and the decrease in the intensity of [ø] showed no simple correlation. The effect of acidic pH (below pH 6–5) on the membrane CD appeared similar to that of increasing salt concentrations. In contrast, the effect of basic pH appeared to be one of denaturation. The effect of temperature on the membrane CD was one of stability below or at physiological temperatures and of irreversible instability at temperatures slightly above the physiological range. The CD of synaptosomal membrane preparations changed slowly over prolonged periods at low temperatures or by cycles of freezing and thawing. Our observations indicate that careful attention to the physical and chemical environment of membranes is necessary in CD investigations of membrane preparations.     

Structure, interaction, dynamics and solvent effects on the DNA-EcoRI complex in aqueous solution from molecular dynamics simulation.

A 0.7-ns molecular dynamics simulation of the DNA-EcoRI complex in a 7.0-A solvent shell indicated a stable behavior of the system. No significant evaporation or smearing of the solvent's outer boundary occurred. The structure and the intermolecular interactions were found to be well maintained during the simulation. The interaction pattern in the simulation was found to be very similar to that in the crystal structure. Most of the specific interactions between the DNA and the protein were found to be enhanced in the simulation compared to that in the crystal structure as a result of improved interaction geometry. The nonspecific interactions were found to be stronger than the specific ones. The specific interactions between the N7 atoms of Gua(4) or Ade(5) or Ade(6) and the protein were found to be present over almost the entire time of the simulation, whereas hydrogen bonds involving the amino groups of the Ade(5) and Ade(6) with the protein were found to be relatively weaker, with lower probability and shorter lifetime. The time evolution of the root mean square deviations of the DNA and the protein were highly correlated even at the later part of the simulation, showing the tight binding between them. Several long-lived water bridges were found between the DNA backbone atoms and the protein and also between the two protein monomers, which increased the overall stability of the complex. The two protein monomers were found to interact strongly with each other. The energy of the DNA kink deformation was estimated as approximately 31 kcal/mol.     

Effects of temperature, lipid modification and pH on the mobility of the major proteins of the receptor-rich membranes from Torpedo marmarata

The factors influencing the overall mobility of the major proteins of the acetylcholine receptor-rich membranes from Torpedo marmorata have been investigated by saturation transfer ESR spectroscopy and the lateral distribution of these proteins has been studied by electron microscopy. A spin-labelled derivative of maleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MSL), was used under various conditions of incubation, enabling us to attach it mainly to either an extrinsic protein of 43 kdaltons, or an intrinsic protein (40 kdaltons) bearing the alpha-toxin-binding site. (1) The direct reaction of MSL with the membrane fragments resulted in almost exclusive labelling of the 43 kdalton protein, an extrinsic protein located on the inner face of the receptor-rich membranes. (2) After the free SH groups were blocked with N-ethylmaleimide and the disulfide bridges opened with the reducing agent dithiothreitol, MSL reacted with both the 40 and 43 kdalton proteins (6.0 +/- 0.6 MSL molecules per alpha-toxin-binding site). (3) After the latter labelling procedure membranes were exposed to pH 11, resulting in extraction of the 43 kdalton protein and leaving 2.2 +/- 0.4 MSL molecules per alpha-toxin-binding site; sodium dodecyl sulfate polyacrylamide gel electrophoresis performed with N-[14C] ethylmaleimide suggested that MSL was bound mainly to the 40 kdalton polypeptide chain of the acetylcholine receptor. The following conclusions were made with the native and alkaline-treated membranes: In the native membranes, saturation transfer ESR does not reveal any significant protein rotational diffusion (rotational correlation time tau C greater than 1 ms). Temperature variations and/or lipid modifications obtained by fusion of exogenous lipids and/or cholesterol exchange have little influence on the saturation transfer ESR spectra. Electron microscopy reveals that upon lipid addition, proteins remain in the form of clusters while areas depleted of proteins appear. On the other hand, alkaline treatment strikingly enhances the motion of the MSL-labelled proteins in the membrane (100 less than or equal to tau c less than or equal to 120 microseconds). Furthermore, the rotational diffusion of the MSL-labelled proteins (mainly the 40 kdalton protein) becomes sensitive to temperature, lipid composition and the lipid-to-proteins ratio. Electron microscopy shows that alkaline extraction does not cause large reorganization of the acetylcholine receptor in the plane of the membrane. However, when phospholipids are added to pH 11 treated membranes, a dispersion of the receptor and rosettes is observed. In contrast, cholesterol enrichment of the latter membranes induces clustering of the receptor immobilization as judged by saturation transfer ESR. Upon reassociation of the pH 11 soluble proteins with the alkaline-treated membranes, the restriction of the acetylcholine receptor rotational mobility is also restored (tau c greater than or equal to 1 ms).     

Analysis of oligomeric proteins during unfolding by pH and temperature

During thermal transition and variation of pH, structural properties of 35 proteins and their complexes (bound with substrate and co-factor) were analyzed in detail. During pH alteration, these proteins were shown to have substantial differences in conformations. pH conformers were analyzed in detail. Free energy and other energy parameters were also estimated for these proteins at various pH and temperatures. Detailed structural analysis and binding interfaces of various substrates, inhibitors and cofactor of these proteins were also investigated using docking and molecular dynamic simulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.