Lysozyme In Aqueous Solutions Research Articles

Influence of pressure on the low‐frequency vibrational modes of lysozyme and water: A complementary inelastic neutron scattering and molecular dynamics simulation study

We performed complementary inelastic neutron scattering (INS) experiments and molecular dynamics (MD) simulations to study the influence of pressure on the low-frequency vibrational modes of lysozyme in aqueous solution in the 1 atm-6 kbar range. Increasing pressure induces a high-frequency shift of the low-frequency part (<10 meV = 80 cm(-1)) of the vibrational density of states (VDOS), g(ω), of both lysozyme and water that reveals a stiffening of the interactions ascribed to the reduction of the protein and water volumes. Accordingly, high pressures increase the curvature of the free energy profiles of the protein quasiharmonic vibrational modes. Furthermore, the nonlinear influence of pressure on the g(ω) of lysozyme indicates a change of protein dynamics that reflects the nonlinear pressure dependence of the protein compressibility. An analogous dynamical change is observed for water and stems from the distortion of its tetrahedral structure under pressure. Moreover, our study reveals that the structural, dynamical, and vibrational properties of the hydration water of lysozyme are less sensitive to pressure than those of bulk water, thereby evidencing the strong influence of the protein surface on hydration water.

Salt-induced pattern formation in evaporating droplets of lysozyme solutions

Solute self-organization during evaporation of colloidal sessile droplets has attracted the attention of researchers over the past few decades due to a variety of technological applications. Recently, pattern formation during evaporation of various biofluids has been studied due to potential applications in screening and diagnosis. The complex morphological patterns in the deposit are unique to various disorders and are influenced by various physical mechanisms occurring during evaporation. These complex patterns can be better understood by studying evaporation of model solutions of biological relevance. Here, we examine the general features of pattern formation during sessile droplet evaporation of aqueous lysozyme solutions with varying concentrations of NaCl. Lysozyme is a globular protein found in biological fluids such as tears and saliva. The morphological evolution of the droplet is studied by time-lapse video during evaporation via reflection optical microscopy. The final deposits exhibit an amorphous peripheral ring and interior regions containing crystallites and dendritic forms, dependent on NaCl concentration. Scanning electron microscopy (SEM) images demonstrate the multi-scale hierarchical nature of these structures.

Physico-chemical studies of oppositely charged protein–surfactant system in aqueous solutions: Sodium dodecyl sulphate (SDS)–lysozyme

Sound velocities (v) and densities (d) of sodium dodecyl sulphate (SDS) in various aqueous solutions of lysozyme have been measured over a temperature range of 25–40°C. The data was used to calculate apparent molar volume (ϕv) and apparent molar adiabatic compressibility (ϕk). These parameters have been examined as a function of surfactant concentration, lysozyme concentration and temperature to understand the consequences of interactions between these two components. Surface tension (γ) measurements were also carried out for the same systems using Man Singh Survismeter. The surface tension data have been used further to calculate the interfacial parameters like, maximum surface excess concentration (Γmax), minimum area per molecule (Amin), standard free energy of adsorption (ΔGad°), surface pressure at CMC (Πcmc) and standard free energy of transfer (ΔGtr°), that have direct bearing on the consequences of such interactions at the molecular level. The work has been extended to include UV-studies for different SDS–lysozyme solutions. A reasonably good qualitative correlation is found to exist with regard to the surfactant–lysozyme interaction obtained from the experimental data, from different techniques.

Small-Angle Neutron Scattering Study of Protein Crowding in Liquid and Solid Phases: Lysozyme in Aqueous Solution, Frozen Solution, and Carbohydrate Powders

The structure, interactions, and interprotein configurations of the protein lysozyme were studied in a variety of phases. These properties have been studied under a variety of solution conditions before, during, and after freezing and after freeze-drying in the presence of glucose and trehalose. Contrast variation experiments have also been performed to determine which features of the scattering in the frozen solutions are from the protein and which are from the ice structure. Data from lysozyme at concentrations ranging from 1 to 100 mg/mL in solution and water ice with NaCl concentrations ranging from 0 to 0.4 mol/L are fit to model small-angle neutron scattering (SANS) intensity functions consisting of an ellipsoidal form factor and either a screened-Coulomb or hard-sphere structure factor. Parameters such as protein volume fraction and long dimension are followed as a function of temperature and salt concentration. The SANS results are compared to real space models of concentrated lysozyme solutions at the same volume fractions obtained from Monte Carlo simulations. A cartoon representation of the frozen lysozyme solution in 0 mol/L NaCl is presented based on the SANS and Monte Carlo results, along with those obtained from other complementary methods.

Label-free impedimetric thrombin sensor based on poly(pyrrole-nitrilotriacetic acid)-aptamer film

A label-free and highly sensitive impedimetric aptasensor was developed based on electropolymerized film for the determination of thrombin. The first step is the electrogeneration of a poly(pyrrole-nitrilotriacetic acid) (poly(pyrrole-NTA)) film onto the surface of electrodes followed by complexation of Cu2+ ions. Then, the histidine labeled thrombin aptamer was immobilized onto the electrode through coordination of the histidine groups on the NTA–Cu2+ complex. The aptamer sensor was applied for the detection and quantification of thrombin via impedimetric detection without a labeling step. A linear quantification of thrombin was obtained in the range 4.7×10–12–5.0×10−10molL−1 with a sensitivity of 2838Ω/log unit (R2=0.9984). The impedance modulus at 0.3Hz as a function of thrombin concentration was used to elaborate a similar linear relationship from 4.7×10–12 to 5×10−10molL−1. In addition, aptamer-poly(pyrrole-NTA) electrodes incubated for 40min in aqueous solutions of bovine serum albumin (BSA), lysozyme and IgG (5×10−7molL−1) did not exhibit non-specific adsorption of proteins. Moreover, it has been demonstrated that the selective sensor can be regenerated several times with a good reproducibility.

Effects of Mannose, Fructose, and Fucose on the Structure, Stability, and Hydration of Lysozyme in Aqueous Solution

The bio-protective properties of monosaccharaides, namely mannose, fructose and fucose, on the stability and dynamical properties of the NMR determined hen egg-white lysozyme structure have been investigated by means of molecular dynamics simulations at room temperature in aqueous solution and in 7 and 13 wt % concentrations of the three sugars. Results are discussed in the framework of the bio-protective phenomena. The three sugars show similar bio-protective behaviours at room temperature (300 K) in the concentration range studied as shown by the small RMSDs of the resulting MD structures from that of starting NMR structure. The effects of sugars on protein conformation are found to be relatively strong in that the conformation of lysozyme is stable after an initial 9 ns equilibration for fucose and mannose and 12 ns equilibration for fructose, respectively, at high concentrations. For mannose the final RMSD is significantly smaller than that of fucose and fructose at the higher concentration, while at the lower concentration the RMSD are essentially the same. The radial distribution function of the water and sugars around lysozyme was used to monitor the preferential hydration. Analysis of the solvent and sugar distributions around lysozyme was used to investigate the interfacial solvent and sugar structure near the protein surface.

A calorimetric study of the interactions in the aqueous solutions of lysozyme in the presence of denaturing cosolvents

A thermodynamic method is reported to monitor the chemical denaturation of lysozyme. Heats of dilution of the protein in concentrated aqueous solutions of urea or ethanol have been determined at 298.15 K by flow microcalorimetry. The pairwise enthalpic interaction coefficients of the protein in the different solvent media are derived. These parameters allow to gain information about the influence of the cosolvents on the interactions acting between two interacting hydrated molecules of lysozyme, hence on the denaturation process. At increasing urea concentration, up to about 6 mol kg−1, the values of the interaction coefficients are large and negative and remain almost unaltered. The invariance of the coefficients underlines that, even in highly concentrated urea, the hydration shell of the protein is such to maintain essentially unaltered the native conformation. At higher urea concentrations, a sudden change in the sign of the coefficients monitors the variation in the interactions between two hydrated denatured protein molecules.The same trend is found when ethanol is the cosolvent. At increasing concentration of the cosolvent, coefficients are, at first, almost invariant. After that, denaturation occurs, detected as a jump toward much more negative values. The results obtained are rationalized on the basis of those previously found for small model molecules in concentrated solutions of urea or ethanol. The thermodynamic framework allows useful comments to be made on the possible mode of action of the two cosolvents on the stability of proteins in solution.

Synthesis of HgS nanocrystals in the Lysozyme aqueous solution through biomimetic method

In the present work, it is reported for Lysozyme-conjugated HgS nanocrystals with tunable sizes prepared at Lysozyme (Lyso) aqueous solutions by using biomimetic method. The obtained HgS nanoparticles with good dispersibility have been characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution transmission microscopy (HRTEM) and energy-dispersive X-ray spectrum (EDS). The Lysozyme molecules can control nucleation and growth of HgS crystals by binding on the surface of nanocrystals to stabilize protein-capped nanoparticles. Quantum confinement effect of Lyso-conjugated HgS nanocrystals has been confirmed by UV–vis spectra. The nanoparticles exhibit a well-defined emission feature at about 470nm. Fourier transform infrared (FT-IR) data are used to envisage the binding of nanoparticles with functional groups of Lysozyme. The results of circular dichroism (CD) spectra indicated that the formation of HgS nanocrystals can lead to conformational change of Lysozyme.

Polar solvation dynamics of lysozyme from molecular dynamics studies

The solvation dynamics of a protein are believed to be sensitive to its secondary structures. We have explored such sensitivity in this article by performing room temperature molecular dynamics simulation of an aqueous solution of lysozyme. Nonuniform long-time relaxation patterns of the solvation time correlation function for different segments of the protein have been observed. It is found that relatively slower long-time solvation components of the α-helices and β-sheets of the protein are correlated with lower exposure of their polar probe residues to bulk solvent and hence stronger interactions with the dynamically restricted surface water molecules. These findings can be verified by appropriate experimental studies.

Relaxation dynamics of a protein solution investigated by dielectric spectroscopy

In the present work, we provide a dielectric study on two differently concentrated aqueous lysozyme solutions in the frequency range from 1MHz to 40GHz and for temperatures from 275 to 330K. We analyze the three dispersion regions, commonly found in protein solutions, usually termed β-, γ-, and δ-relaxations. The β-relaxation, occurring in the frequency range around 10MHz and the γ-relaxation around 20GHz (at room temperature) can be attributed to the rotation of the polar protein molecules in their aqueous medium and the reorientational motion of the free water molecules, respectively. The nature of the δ-relaxation, which is often ascribed to the motion of bound water molecules, is not yet fully understood. Here we provide data on the temperature dependence of the relaxation times and relaxation strengths of all three detected processes and on the dc conductivity arising from ionic charge transport. The temperature dependences of the β- and γ-relaxations are closely correlated. We found a significant temperature dependence of the dipole moment of the protein, indicating conformational changes. Moreover we find a breakdown of the Debye–Stokes–Einstein relation in this protein solution, i.e., the dc conductivity is not completely governed by the mobility of the solvent molecules. Instead it seems that the dc conductivity is closely connected to the hydration shell dynamics.

Biomimetic synthesis and characterisation of ZnS nanoparticles in aqueous solution of lysozyme

Zinc sulphide nanoparticles were prepared by a simple biomolecule-assisted biomimetic method at room temperature. X-ray diffraction pattern confirmed the formation of a cubic zinc blende phase of ZnS nanocrystals. Transmission electron microscopy images revealed that the nanoparticles are nearly spherical in shape, with average diameter about 40 nm. The optical property of ZnS nanoparticles was studied by the ultraviolet–visible spectrum. Different template concentrations of lysozyme are used to investigate the influence of biomolecule on the formation of nanoparticles. Fourier transform infrared spectroscopy study suggested ZnS nanoparticles can react with functional groups of lysozyme including –OH and –NH.

Effective interactions in lysozyme aqueous solutions: A small-angle neutron scattering and computer simulation study

We report protein-protein structure factors of aqueous lysozyme solutions at different pH and ionic strengths, as determined by small-angle neutron scattering experiments. The observed upturn of the structure factor at small wavevectors, as the pH increases, marks a crossover between two different regimes, one dominated by repulsive forces, and another one where attractive interactions become prominent, with the ensuing development of enhanced density fluctuations. In order to rationalize such experimental outcome from a microscopic viewpoint, we have carried out extensive simulations of different coarse-grained models. We have first studied a model in which macromolecules are described as soft spheres interacting through an attractive r(-6) potential, plus embedded pH-dependent discrete charges; we show that the uprise undergone by the structure factor is qualitatively predicted. We have then studied a Derjaguin-Landau-Verwey-Overbeek (DLVO) model, in which only central interactions are advocated; we demonstrate that this model leads to a protein-rich/protein-poor coexistence curve that agrees quite well with the experimental counterpart; experimental correlations are instead reproduced only at low pH and ionic strengths. We have finally investigated a third, "mixed" model in which the central attractive term of the DLVO potential is imported within the distributed-charge approach; it turns out that the different balance of interactions, with a much shorter-range attractive contribution, leads in this latter case to an improved agreement with the experimental crossover. We discuss the relationship between experimental correlations, phase coexistence, and features of effective interactions, as well as possible paths toward a quantitative prediction of structural properties of real lysozyme solutions.

1PT162 リゾチームの乾燥誘導凝集に対するG3LEAタンパク質及びモデルペプチドの抑制効果(日本生物物理学会第50回年会(2012年度))

1PT162 リゾチームの乾燥誘導凝集に対するG3LEAタンパク質及びモデルペプチドの抑制効果(日本生物物理学会第50回年会(2012年度))

Characteristics of hydration water around hen egg lysozyme as the protein model in aqueous solution. FTIR spectroscopy and molecular dynamics simulation

In this paper, the hydration of a model protein--hen egg white lysozyme in aqueous solution has been presented. The leading method used was FTIR spectroscopy with an application of a technique of semi-heavy water (HDO) isotope dilution. Analysis of spectra of HDO isotopically diluted in water solution of lysozyme allowed us to isolate HDO spectra affected by lysozyme, and thus to characterise the energetic state of water molecules and their arrangement around protein molecules. The number of water molecules and the shape of the affected HDO spectrum were obtained using a classical and a chemometric method. This shape showed that the HDO spectrum affected by lysozyme may be presented as a superposition of two spectra corresponding to HDO affected by N-methylacetamide and the carboxylate anion (of the formic acid). Moreover, based on the difference in intermolecular distances distribution of water molecules (obtained from spectral data), we demonstrated that the lysozyme molecule causes a decrease in population of weak hydrogen bonds, and concurrently increases the probability of an occurrence of short hydrogen bonds in water affected by lysozyme. This conclusion was also confirmed by the molecular dynamics (MD) simulation.

New routes to food gels and glasses

We describe the possibility to create solid-like protein samples whose structural and mechanical properties can be varied and tailored over an extremely large range in a very controlled way through an arrested spinodal decomposition process. We use aqueous lysozyme solutions as a model globular protein system. A combination of video microscopy, small-angle neutron and X-ray scattering and reverse Monte Carlo modeling is used to characterize the structure of the bicontinuous network with two coexisting phases of a dilute protein solution and a glassy or arrested dense protein backbone at all relevant length scales. Rheological measurements are then used to determine the complex mechanical response of these protein gels as a function of protein concentration and quench temperature. While in particular the origin of the dependence of the mechanical properties on quench depth and concentration is not well understood currently, it seems ultimately connected to the particular bicontinuous structure of the arrested spinodal network created by the interplay between the early stage of a spinodal decomposition and the position of the glass line. We then generalize this behavior and discuss how this could open up new routes to prepare gel-like food systems with adjustable structural and mechanical properties. We present results from a first feasibility study where we use a depletion interaction caused by the addition of small non-adsorbing polymers to suspensions of casein micelles in order to create food gels with tunable structural and mechanical properties through an arrested spinodal decomposition process.

Local heterogeneous dynamics of water around lysozyme: a computer simulation study

Water present near the surface of a protein exhibits dynamic properties different from that of water in the pure bulk state. In this work, we have carried out atomistic molecular dynamics simulation of an aqueous solution of hen egg-white lysozyme. Attempts have been made to explore the correlation between the local heterogeneous mobility of water around the protein segments and the rigidity of the hydration layers with the microscopic dynamics of hydrogen bonds formed by water molecules with the protein residues. The kinetics of breaking and reformation of hydrogen bonds involving the surface water molecules have been calculated. It is found that the reformations of broken hydrogen bonds are more frequent for the hydration layers of those segments of the protein that are more rigid. The calculation of the low-frequency vibrational modes of hydration layer water molecules reveals that the protein influences the transverse and longitudinal degrees of freedom of water around it in a differential manner. These findings can be verified by appropriate experimental studies.

Viscometric study of lysozyme solution with sugar and urea at various temperatures

This paper presents the results of viscosity measurement of three ternary systems i.e.1-d (−) Glucose+lysozyme+water2-Maltose+lysozyme+water3-Urea+lysozyme+waterat temperatures (293.15, 303.15, 313.13 and 323.15K) at various concentrations of glucose, maltose and urea. Change in entropy (ΔH), enthalpy (ΔS) and free energy of activation (ΔG) have also been evaluated for these systems. Value of B-coefficient of d (−) glucose, maltose and urea has also been calculated from viscosity data in aqueous lysozyme solution. Viscosity B-coefficients of glucose and maltose in aqueous lysozyme solution are positive while that of the urea–lysozyme water system it is negative due to the structure breaking effect of urea. The values of entropy of activation are negative due to attainment of transition state for viscous flow, which is accompanied by bond formation and increase in order.

Molecular properties of lysozyme-microbubbles: towards the protein and nucleic acid delivery

Microbubbles (MBs) have specific acoustic properties that make them useful as contrast agents in ultrasound imaging. The use of the MBs in clinical practice led to the development of more sensitive imaging techniques both in cardiology and radiology. Protein-MBs are typically obtained by dispersing a gas phase in the protein solution and the protein deposited/cross-linked on the gas-liquid interface stabilizes the gas core. Innovative applications of protein-MBs prompt the investigation on the properties of MBs obtained using different proteins that are able to confer them specific properties and functionality. Recently, we have synthesized stable air-filled lysozyme-MBs (LysMBs) using high-intensity ultrasound-induced emulsification of a partly reduced lysozyme in aqueous solutions. The stability of LysMBs suspension allows for post-synthetic modification of MBs surface. In the present work, the protein folded state and the biodegradability property of LysMBs were investigated by limited proteolysis. Moreover, LysMBs were coated and functionalized with a number of biomacromolecules (proteins, polysaccharides, nucleic acids). Remarkably, LysMBs show a high DNA-binding ability and protective effects of the nucleic acids from nucleases and, further, the ability to transform the bacteria cells. These results highlight on the possibility of using LysMBs for delivery of proteins and nucleic acids in prophylactic and therapeutic applications.

Effect of Lysozyme Proteins on the Mutual-Diffusion Coefficient of Sodium Chloride in Water

We measured accurate NaCl main-term diffusion coefficients in aqueous lysozyme solutions at 25 °C and pH 4.5 using the Gosting diffusiometer operated in its Rayleigh interferometric optical mode. The dependence of this diffusion coefficient on lysozyme concentration was examined using the obstruction-effect theory. Agreement between experimental results and theory is achieved if lysozyme proteins are treated as hydrated spheres with a hydration number of 240, a value that is comparable with those reported in literature for this protein. Electrostatic interactions and common-ion effects due to lysozyme net charge at pH 4.5 do not contribute significantly to the behavior of the NaCl diffusion coefficient within our experimental range of salt concentrations [(0.25 to 0.90) mol·dm–3].

Effect of PEG End-Group Hydrophobicity on Lysozyme Interactions in Solution Characterized by Light Scattering

We compare protein-protein and protein-polymer osmotic virial coefficients measured by static light scattering for aqueous solutions of lysozyme with low-molecular-weight, hydroxy-terminated (hPEG) and methyl-terminated (mPEG) poly(ethylene glycol) at two solution conditions: pH 7.0 and 0.01 M ionic strength, and pH 6.2 and 0.8 M ionic strength. We find that adding PEG to aqueous lysozyme solutions makes a net repulsive contribution to lysozyme-lysozyme interactions, independent of ionic strength and PEG end-group hydrophobicity. PEG end-group hydrophobicity has a profound effect on the magnitude of this contribution, however, at low ionic strength where mPEG-lysozyme attractive interactions become significant. The enhanced attractions promote mPEG-lysozyme preferential interactions at the expense of lysozyme self-interactions, which leads to lysozyme-lysozyme interactions that are more repulsive in the presence of mPEG. These preferential interactions also lead to the preferential exclusion of diffusable ions locally around the protein, which results in a pronounced ionic strength dependence of mPEG-mediated lysozyme-lysozyme interactions.