Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride

Recent studies have outlined the use of eutectic solutions of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without solvent crystallization. The eutectic point in a (H₂O)_R(LiCl) system corresponds to R ≈ 7.3, and it is of interest to investigate whether less‐concentrated aqueous solutions of LiCl could be used in low‐temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low‐temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 624–629, 2014.     

Ion solvation and water structure in potassium halide aqueous solutions

The structure of water and the nature of ionic hydration is explored in aqueous solutions of potassium fluoride, chloride, bromide and iodide over a range of concentrations up to 4.8 ion pairs per 100 water molecules, using the combined techniques of neutron diffraction with hydrogen isotope substitution. The diffraction data are interpreted using the method of empirical potential structure refinement, which attempts to build a three-dimensional model of the scattering system consistent with the diffraction data. The water structure is strongly perturbed in the first hydration shells of both anion and cation, but is found to be only mildly perturbed outside of this region, with the largest effects occurring with the smallest anion and highest concentrations. For the potassium ion there are strong orientational correlations in the first hydration shell, with the water molecules lying with their dipole moments pointing almost directly away from the cation on average, but with an angular spread of approximately +/-60 degrees which is mildly dependent on the anion type present. For all the anions the water molecules in the first shell are strongly oriented with one O-H vector pointing directly towards the anion on average, with an angular spread of approximately +/-10 degrees for F(-), increasing to approximately +/-22 degrees for I(-). For both anions and cations the second hydration shell is much more disordered than the first, but there is a weak pattern of orientational correlation which becomes more pronounced with the larger anions. There is some evidence that the fluoride ion structures water significantly in its first hydration shell, but not beyond. The findings throw further light on recent findings that the orientational relaxation time for water outside the first shell of dissolved ions is the same as in the bulk liquid.     

Pressure-induced unfolding of lysozyme in aqueous guanidinium chloride solution.

The pressure-induced unfolding of lysozyme was investigated in an aqueous guanidinium chloride solution by means of ultraviolet spectroscopy. Assuming a two-state transition model, volume changes were calculated from the slope of free energy vs. pressure plots over a temperature range of 10 to 60 degrees C. Between 25 and 60 degrees C, almost constant volume changes were observed in the transition region, which was reflected in almost identical slopes of the free energy change vs. pressure plots. On the other hand, the different slopes were observed in the pressure dependence of free energy change at temperatures lower than 25 degrees C. These data were interpreted as suggesting that a two-state model is not appropriate at low temperature, but instead one or more intermediates are present under these conditions. The volume changes for unfolding became less negative at temperatures higher than 25 degrees C.     

Electroviscous effect of lysozyme in aqueous solutions

Reduced viscosity of a dilute aqueous solution of hen egg white lysozyme is measured in the pH range from 1.4 to 12.7 for various NaCI concentrations. The viscosity decreases with increasing pH below the isoelectric point (pH 11) on account of diminution in the electroviscous effect, reaches a minimum at pH 11, and then increases at high pH's because of coagulation. The electroviscous effect is depressed by the increase in the small ion concentration. The dependence of reduced viscosity on small ion concentration and pH is discussed on the basis of Booth's theory and a partial agreement between theory and experiment is obtained. The discrepancy between theory and experiment is attributed to non-spherical distribution of charges in the protein. The volume of lysozyme obtained through Einstein's equation by extrapolating the reduced viscosity to a sufficiently high ion concentration compares well with the molecular volume in the crystal.     

Interactions of lysozyme in guanidinium chloride solutions from static and dynamic light-scattering measurements

The interactions of partially unfolded proteins provide insight into protein folding and protein aggregation. In this work, we studied partially unfolded hen egg lysozyme interactions in solutions containing up to 7 M guanidinium chloride (GdnHCl). The osmotic second virial coefficient (B(22)) of lysozyme was measured using static light scattering in GdnHCl aqueous solutions at 20 degrees C and pH 4.5. B(22) is positive in all solutions, indicating repulsive protein-protein interactions. At low GdnHCl concentrations, B(22) decreases with rising ionic strength: in the absence of GdnHCl, B(22) is 1.1 x 10(-3) mLmol/g(2), decreasing to 3.0 x 10(-5) mLmol/g(2) in the presence of 1 M GdnHCl. Lysozyme unfolds in solutions at GdnHCl concentrations higher than 3 M. Under such conditions, B(22) increases with ionic strength, reaching 8.0 x 10(-4) mLmol/g(2) at 6.5 M GdnHCl. Protein-protein hydrodynamic interactions were evaluated from concentration-dependent diffusivity measurements, obtained from dynamic light scattering. At moderate GdnHCl concentrations, lysozyme interparticle interactions are least repulsive and hydrodynamic interactions are least attractive. The lysozyme hydrodynamic radius was calculated from infinite-dilution diffusivity and did not change significantly during protein unfolding. Our results contribute toward better understanding of protein interactions of partially unfolded states in the presence of a denaturant; they may be helpful for the design of protein refolding processes that avoid protein aggregation.     

Effects of surfactants on emulsification and secondary structure of lysozyme in aqueous solutions

This paper reports our investigations on the secondary structure of lysozyme in aqueous solution with D₂O and comparing systems emulsified with ionic and non-ionic amphiphiles, respectively. The opposite effect of concentration of surfactants on the lysozyme aqueous system was studied and the activity of lysozyme by the turbidimetric assay with different kinds of surfactant was compared. The effect of different surfactants on emulsification volume formed was observed and discussed. The different interactive patterns between lysozyme and ionic and non-ionic surfactants, such as sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB) and Triton-X100 (Isooctoylphenoxypolyethoxy ethanol), respectively, was studied with FTIR. A simple relationship between the secondary structure and denaturation of protein is given for the emulsification of lysozyme in aqueous solutions. It is important for understanding the stabilization and avoiding the emulsions in the extraction process that the interactions between the proteins and surfactants which leads to the formation of such complexes are characterized. The utility of surfactants as a de-emulsifying agent for the commercial process to extract penicillin G from the filtrate of fermentation broth was consistent with these results.     

Multiple conformational transitions of hen egg-white lysozyme in aqueous acetic acid solutions

Circular dichroism measurements revealed that hen egg-white lysozyme underwent multiple conformational transitions upon the addition of acetic acid. The transitions were reversible as judged from complete recoveries of enzymatic activity, electrophoretic mobility in SDS-polyacrylamide gel, and of ellipticity. Two transitions, with the mid-concentrations of 26 and 38% (v/v), were observed with the CD spectra in the amide absorption region. The two transitions were essentially athermal in the temperature ranges, 0 to 25 degrees C for the former and -10 to 10 degrees C for the latter. The trough ellipticity for the product of the transition at the higher acetic acid concentration (DII form) very closely approached the value for the synthetic polypeptides in the beta-conformation as the temperature was lowered. Molecular weight measurements by sedimentation equilibrium indicated that the products were both monomeric. Measurements of CD spectra in the aromatic absorption region showed another transition, whose mid-concentration varied with temperature from 26% (v/v) (at about 25 degrees C) to 38% (v/v) (at -10 degrees C). A change in the hydrodynamic volume detectable by exclusion chromatography was associated with this transition only.     

Dielectric behavior of lysozyme and ferricytochrome-c in water/ethylene-glycol solutions

This work deals with a dielectric study at radio frequencies of the influence at room temperature of two organic molecules, known as cryo-protectants, ethylene-glycol and glycerol, on conformational and dynamic properties of two model proteins, lysozyme (lys) from chicken egg-white and ferricytochrome-c (cyt-c) from horse heart. Cyt-c is a compact globular protein whereas lys is composed of two structural domains, separated by the active site cleft. Measurements were carried out at the fixed temperature of 20 degrees C varying the concentration of the cosolvent up to 90% w/w. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius and the electric dipole moment of the protein were calculated as a function of the cosolvent concentration. The data show that glycerol does not modify significantly the conformation of both proteins and cyt-c is also stable in the presence of ethylene-glycol. On the contrary ethylene-glycol strongly affects the dielectric response of lysozyme denoting a specific effect on its conformation and dynamics. The data are coherently interpreted hypothesizing that glycol molecule wedges between and separates the two domains of lys making them rotationally independent.     

Conformation of xanthan dissolved in aqueous urea and sodium chloride solutions

Quasielastic light-scattering and other physical-chemical techniques have been used to compare the conformation and intermolecular interactions of xanthan in water, aqueous sodium chloride, and urea solutions. The results showed that xanthan dissolved in 4m urea has a disordered conformation after the solution has been maintained for 3 h at 95° and then cooled to room temperature. This conformation is similar to that previously observed only in solutions having low ionic strength at higher temperatures, following disruption of the ordered, low-temperature form. “Anomalous” behavior is seen for xanthan as a function of ionic strength, in that the hydrodynamic radius increases with increase in ionic strength, whereas a decrease is typical for polyelectrolytes. These observations suggest that aggregation of rod-like chains, similar to that seen for other stiff-chain polymers, occurs for xanthan in salt solutions, where the charged groups of the polyelectrolyte are screened by the salt ions. This aggregation may explain some of the high values reported in the literature for the molecular weight.     

Ultrasonic absorption in aqueous solution of lysozyme.

The titration curve of ultrasonic absorption at 2.82 MHz in aqueous solutions of lysozyme measured by Zana and Lang [J. Phys. Chem., 74 , 2734 (1970)] is theoretically analyzed. The maxima at pH 3 and pH 11 are describable with proton-transfer reactions of dissociable carboxyl and amino groups by assuming that volume changes due to the reactions are 2.3 and 5.2 cm³/mole, respectively, which are appreciably smaller than those of simple amino acids. The remaining, pH-independent excess absorption over solvent is measured at frequencies ranging from 3 to 150 MHz. The absorption is ascribed to the internal loss of protein. The complex compressibility β′_p ? iβ″_p of lysozyme molecule is evaluated as β′_p = 7.2 × 10^?12 cm²/dyne and β″_P = 4.3 × 10^?14 cm²/dyne from the increments over solvent in absorption as well as in sound velocity.     

Mid-infrared spectroscopic analysis of saccharides in aqueous solutions with sodium chloride

The infrared spectral characteristics of three different types of disaccharides (trehalose, maltose, and sucrose) and four different types of monosaccharides (glucose, mannose, galactose, and fructose) in aqueous solutions with sodium chloride (NaCl) were determined. The infrared spectra were obtained using the FT-IR/ATR method and the absorption intensities respected the interaction between the saccharide and water with NaCl were determined. This study also focused on not only the glycosidic linkage position and the constituent monosaccharides, but also the concentration of the saccharides and NaCl and found that they have a significant influence on the infrared spectroscopic characterization of the disaccharides in an aqueous solution with NaCl. The absorption intensities representing the interaction between a saccharide and water with NaCl were spectroscopically determined. Additionally, the applications of MIR spectroscopy to obtain information about saccharide–NaCl interactions in foods and biosystems were suggested.     

Sodium chloride, potassium chloride, and virulence in Listeria monocytogenes.

Virulence, as determined in a mouse model, and the virulence factor activities of catalase, superoxide dismutase, and listeriolysin O were examined in a parental strain (10403S) and in a nonhemolytic mutant strain (DP-L224) of Listeria monocytogenes. The cells were propagated in media containing various concentrations of sodium chloride or potassium chloride. Strains 10403S and DP-L224 exhibited significant increases in catalase activity and listeriolysin O activity when grown in medium containing either salt at 428 mM. The superoxide dismutase activities for both strains increased when they were grown in medium containing either salt. The superoxide dismutase activity was significantly increased only when cells were propagated in medium containing no salt compared with that when they were propagated in medium containing either salt at 1,112 mM. In addition, the listeriolysin O activity was highest for cells propagated in medium containing KCl at 428 mM, while the activity was significantly less for cells propagated in medium containing NaCl at an equal concentration. Virulence was examined in mouse livers and spleens after intravenous infection, and approximate 50% lethal doses were determined after intragastric and intraperitoneal infection. Each method of infection indicated that listeriolysin O is required for virulence, while growth in salt-containing medium or the production of higher levels of catalase, superoxide dismutase, and listeriolysin O do not appear to enhance the virulence of L. monocytogenes.     

Oxidation of tryptophan in lysozyme by ozone in aqueous solution.

A tryptophan residue in hen's egg-white lysozyme [EC 3.2.1.17] was modified by ozone in an aqueous solution. One of the six tryptophan residues in the enzyme was oxidized to N'-formylkynurenine with concomitant loss of the enzymatic activity. Physicochemical studies of this modified enzyme (OL-I) revealed that the ozonization of lysozyme in aqueous media resulted in little change of the gross molecular conformation. It was deduced that the modified tryptophan residue in OL-I was possibly located in position 62 (or 63) of the protein.     

Diffusion in immobilized-cell agar layers: influence of bacterial growth on the diffusivity of potassium chloride

Summary The diffusitivity of potassium chloride in composite agar slab/microporous membrane structures loaded with various amounts of Escherichia coli whole cells was determined using both time-lag and steady-state methods. The diffusion coefficient of KCl decreased linearly with the logarithm of the immobilized-cells content. The effect exerted by bacterial growth inside the immobilization matrices on KCl diffusivity was then investigated. The diffusion coefficient of KCl obtained by time-lag analysis decreased during incubation of the immobilized-cell structures, whereas less consitent results arose from the steady-state method. An apparent doubling time for immobilized E. coli, increasing with the initial cell content of the gel, was obtained from the calibration relationship between KCl diffusivity and the number of organisms in agar. Offprint requests to: G.-A. Junter