State Of Hen Egg-white Lysozyme Research Articles

PH-Dependent HEWL-AuNPs Interactions: Optical Study.

Optical methods (spectroscopy, spectrofluorometry, dynamic light scattering, and refractometry) were used to study the change in the state of hen egg-white lysozyme (HEWL), protein molecules, and gold nanoparticles (AuNPs) in aqueous colloids with changes in pH, and the interaction of protein molecules with nanoparticles was also studied. It was shown that changing pH may be the easiest way to control the protein corona on gold nanoparticles. In a colloid of nanoparticles, both in the presence and absence of protein, aggregation-deaggregation, and in a protein colloid, monomerization-dimerization-aggregation are the main processes when pH is changed. A specific point at pH 7.5, where a transition of the colloidal system from one state to another is observed, has been found using all the optical methods mentioned. It has been shown that gold nanoparticles can stabilize HEWL protein molecules at alkaline pH while maintaining enzymatic activity, which can be used in practice. The data obtained in this manuscript allow for the state of HEWL colloids and gold nanoparticles to be monitored using one or two simple and accessible optical methods.

Study on Irradiation Effect of Mid-Infrared Free Electron Laser on Hen Egg-White Lysozyme by Using Terahertz-Time Domain Spectroscopy and Synchrotron-Radiation Vacuum-Ultraviolet Circular-Dichroism Spectroscopy

Mid-infrared free-electron laser (MIR-FEL) is potentially applicable for tissue ablation and dissociation of pathological peptide aggregates in medicine. However, it is still poorly understood how the MIR-FEL irradiation influences on functional proteins such as enzymes. In the current study, the effect of MIR-FEL on both aggregate and non-aggregate (= native) states of hen egg-white lysozyme (HEWL) as a representative enzyme has been investigated. Absorption intensity at terahertz region (0.3–1.2 THz, 10–40 cm−1) of the aggregate of HEWL was lower than that of the native HEWL, but the former was increased nearly to the same level with the latter after the MIR-FEL irradiation tuned to 6 μm that corresponds to carbonyl stretching vibrational mode of amide bonds (amide I). This indicates that the aggregate of HEWL was converted to the native state by the irradiation. On the other hand, synchrotron-radiation vacuum-ultraviolet circular-dichroism spectroscopy showed that protein conformation of the native HEWL, which was rich in α-helix, was little changed after the MIR-FEL irradiation under the same condition with the case of the aggregate of HEWL. Furthermore, the enzymatic hydrolysis activity of the native HEWL against bacterial glycan was not remarkably decreased by the irradiation. Therefore, it can be estimated that the native structure of HEWL is little damaged although the aggregate state can be easily dissociated by the resonant excitation at amide bonds.

Identification and Characterization of Molten Globule-Like State of Hen Egg-White Lysozyme in Presence of Salts Under Alkaline Conditions

In the present study, we elucidated the effect of potassium salts on alkali denatured hen egg white lysozyme (EC 3.2.1.17) using intrinsic/extrinsic fluorescence as well circular dichroism (CD) spectroscopic methods. Intrinsic fluorescence studies revealed that various potassium salts mediate stabilization of lysozyme against alkali denaturation. Far and near UV CD spectrum studies, showed that 2M KCl induced appreciable amount of secondary structure with minimum tertiary contacts in lysozyme at pH 12.6. Acrylamide quenching studies suggest that at pH 12.6, the presence of 2M KCl causes reduced accessibility of the quencher to tryptophan residues of the protein presumably because of its compact conformation. In summary, the results of present study suggest that lysozyme attains a compact folded intermediate with molten globule like characteristics at alkaline pH in presence of potassium chloride.

Different Molten Globule-like Folding Intermediates of Hen Egg White Lysozyme Induced by High pH and Tertiary Butanol

We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.

Observation of phase states of hen egg-white lysozyme using pressure as a probe

Observation of phase states of hen egg-white lysozyme using pressure as a probe

Effects of organic solvents on protein structures: observation of a structured helical core in hen egg-white lysozyme in aqueous dimethylsulfoxide.

A partly folded state of hen egg-white lysozyme has been characterized in 50% DMSO. Low concentrations of DMSO (< 10%) have little effect on the overall folded conformation of lysozyme as seen from 1H NMR chemical shift dispersion. At increasing DMSO concentrations (> 10%) a cooperative transition of the structure to a new, partially folded state is observed. This transition is essentially complete by approximately 50% DMSO. NMR studies show an overall decrease in chemical shift dispersion with marked broadening of many resonances. A substantial number of backbone and side chain-side chain NOEs suggests the presence of secondary and tertiary interactions in the intermediate state. Tertiary organization of the aromatic residues is also demonstrated by enhanced near-UV circular dichroism and limited exposure of tryptophans as monitored by iodide quenching of fluorescence. The intermediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural transition from this state to a largely unfolded conformation is cooperative. H/D exchange rates of several amide protons and four indole protons of tryptophans (W28, W108, W111, and W123), measured by refolding from 50% DMSO at different time intervals reveal that protection factors are high for the helical domain, whereas NH groups in the triple stranded antiparallel beta-sheet domain are largely solvent-exposed. An ordered hydrophobic core in the intermediate state comprising of helix A, helix B, and helix D is consistent with the high protection factors observed. The structured intermediate in 50% DMSO resembles the early kinetic intermediate observed in the refolding of hen egg white lysozyme, as well as a molten globule state of equine lysozyme at low pH. The results demonstrate the potential use of non-aqueous structure perturbing solvents like DMSO to stabilize partially folded conformations of proteins.

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

Ionic‐Strength‐Dependent Transition of Hen Egg‐White Lysozyme at Low PH to a Compact State and its Aggregation on Thermal Denaturation

Equilibrium acid-induced unfolding of hen egg-white lysozyme has been investigated by a combination of optical methods, size-exclusion chromatography, and differential scanning calorimetry. The results showed the presence of a partially folded state of hen egg-white lysozyme at pH 1.5, characterized by a substantial secondary structure, a large solvent exposure of non-polar clusters, and significantly disrupted tertiary structure. A large enthalpy was also associated with the conversion of the acid-unfolded state to a fully unfolded state. Size-exclusion chromatography and 8-anilino-1-naphthalenesulphonic acid-binding studies showed an ionic-strength-induced transition of the partially folded state to a compact conformation. Furthermore, an ionic-strength-dependent aggregation on thermal unfolding of the partially folded intermediate was also observed. These observations provide insights into the possible features responsible for the stabilization of intermediates in the folding of hen egg-white lysozyme.

PH-Dependence of Protein Stability: Absolute Electrostatic Free Energy Differences between Conformations

A method for calculating the absolute electrostatic free energy of a titrating system as a function of pH is proposed, and a concise formula for the free energy is presented. Based on the theory of linked functions, the electrostatic free energy is calculated by integration of the titration curve. The approach uses pH = ∞, at which the system is in the unprotonated state, as the reference pH for the integration. The finite-difference Poisson−Boltzmann method is used for the electrostatic free energy, and the titration curve is obtained by the Monte Carlo approach of Beroza et al.1 The method is applied to the native and denatured state of hen egg-white lysozyme in aqueous solution. A dielectric constant of 20 is assigned to the protein interior, in accord with the work of Antosiewicz et al.2 X-ray structures are used for the native state, and an extended β-structure is used for the unfolded reference state; good agreement with experiment is obtained. Comparison of the results for the extended β-structure ...