Chicken Egg White Lysozyme Research Articles

Azo-dyes based small bifunctional molecules for metal chelation and controlling amyloid formation.

Chemical tools are needed to discover new effective drugs for tackling multifaceted complex neurodegenerative diseases like Alzheimer's disease (AD). Multifunctional nature of two compounds, 5-((4-nitro-phenyl)diazenyl)quinolin-8-ol (HL1) and 4-((4-nitrophenyl)diazenyl)benzene-1,3-diol (HL2) is reported w.r.t. their ability to bind Cu2+ ions and amyloid aggregates related to AD. HL1 and HL2 have half congo-red type azo-stilbene structural framework incorporated with metal chelating groups, designed to chelate metal ions from metal-amyloid species. Metal binding studies of HL1 and HL2 are established by the methods of Job's Plot, UV-vis spectra with metal ions and stability constant determination. In addition, their metal complexes are isolated, purity checked by elemental analysis, spectroscopically characterized and their structural analyses were obtained from DFT based calculations including binding energy determination. Chicken egg white Lysozyme (CEWL) was used as a model peptide for fibrillation studies. HL1 is found as an excellent colorimetric sensor for amyloid fibrils. Inhibitory effect of HL1 and HL2 and their isolated metal complexes L1-Cu and L2-Cu on CEWL fibrillation was studied using ThT and ANS fluorescence assay along with TEM imaging. In addition, the cell toxicity studies on these compounds suggest that although azo dyes may be non-toxic but having a nitro-substitution lead to significant cell toxicity. Overall, these results suggest that this new class of multifunctional small molecules can interact with amyloids as well as metal ions and could be potential anti-aggregation metal chelating agents.

Fluorescence Behavior of Schiff Base-N, N'-bis(salicylidene) Trans 1, 2-Diaminocyclohexane in Proteinous and Micellar Environments.

Fluorescence properties of N, N'-bis(salicylidene) trans 1, 2-diaminocyclohexane (H 2 L) is used to probe the anionic (SDS), cationic (CTAB) and nonionic (TX-100) micelles as well as in serum albumins (BSA and HSA) and chicken egg white lysozyme (LYZ) by steady state and picosecond time-resolved fluorescence spectroscopy. The fluorescence band intensity was found to increase with concomitant blue-shift with gradual addition of different surfactants. All the experimental results suggest that the probe molecule resides in the micelle-water interface rather than going into the micellar core. However, the penetration is more towards the micellar hydrocarbon core in nonionic surfactant (TX-100) while comparing with ionic surfactants (SDS and CTAB). Several mean microscopic properties such as critical micelle concentration, polarity parameters and binding constant were calculated in presence of different surfactants. The decrease in nonradiative decay rate constants in micellar environments indicates restricted motion of the probe inside the micellar nanocages with increasing fluorescence emission intensity and quantum yields. Further in this work, we also investigated the interaction behavior of the probe with different proteins at low concentrations under physiological conditions (pH = 7.4). Stern-Volmer analysis of the tryptophan (Trp) fluorescence quenching data in presence of probe reveals Stern-Volmer constant (Ksv) as well as bimolecular quenching rate constant (Kq). The binding constant as well as the number of binding sites of the probe with proteins were also monitored and found to be 1:1 stoichiometry ratio.

Specific Affinity Enrichment of Electrochemically Cleaved Peptides Based on Cu(II)-Mediated Spirolactone Tagging.

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) residues provides a potential alternative to enzymatic digestion and a possibility for further chemical labeling by introducing reactive spirolactone moieties. However, spirolactone-containing peptides suffer from low stability due to hydrolysis and intramolecular side reactions. We found that Cu(II) ions stabilize the spirolactone and prevent intramolecular side reactions during chemical labeling, allowing efficient chemical tagging with a reduced excess of labeling reagent without intramolecular side reactions. On the basis of this reaction, we developed an analytical procedure combining electrochemical digestion, Cu(II)-mediated spirolactone biotinylation, and enrichment by avidin affinity chromatography with mass spectrometry. The method was optimized with the tripeptide LWL and subsequently applied to chicken egg white lysozyme, in which one biotinylated electrochemistry (EC)-cleaved peptide was identified after affinity enrichment. This proof-of-principle shows that specific enrichment of electrochemically cleaved spirolactone-containing peptides can be used for protein identification and notably that inclusion of Cu(II) ions is essential for stabilizing spirolactones for subsequent biotinylation.

Elucidation of Binding Mechanism of Photodynamic Therapeutic Agent Toluidine Blue O with Chicken Egg White Lysozyme by Spectroscopic and Molecular Dynamics Studies.

The nature of binding mechanism of toluidine blue O (TBO) with chicken egg white lysozyme was studied comprehensively by various spectroscopic and computational methods. Both steady state and time-resolved fluorescence studies unambiguously point to the prevalence of static quenching mechanism in lysozyme-TBO system. Thermodynamic parameters revealed that the association of TBO with lysozyme was a spontaneous process in which hydrophobic and hydrogen bond interactions played a pivotal role in the binding process. The secondary and tertiary conformational changes of lysozyme in the presence of TBO were unraveled using absorption, Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD) techniques. Molecular docking studies of lysozyme-TBO system substantiated the findings of site marker experiment and revealed TBO adjacent to Trp-63 and Trp-108 residues of lysozyme. Molecular dynamics (MD) simulation studies of lysozyme-TBO system indicate a stable and effective complexation of TBO with lysozyme. It is hoped that the results presented here will enable further understanding of TBO toxicity.

Solid-liquid equilibrium for proteins in solutions with an unconventional salt (ammonium carbamate): Phase behavior analysis

In this manuscript, we report the precipitation and crystallization of three proteins (chicken egg-white lysozyme, porcine insulin and bovine insulin) using ammonium carbamate as salting-out agent. For these proteins, data on solubility, metastability limits, and osmotic second virial coefficient (B22) as a function of salt concentration at constant temperature (25.0 °C) were obtained. The value of osmotic second virial coefficient (B22) can be regarded as a selection criterion for protein crystallization, as it is related to the interaction potential between protein molecules. Negative values of B22 and large values of the salting-out constant showed that ammonium carbamate is a good precipitating agent for these proteins. Crystallization trials conducted under specific conditions showed that both insulins form amorphous precipitates in ammonium carbamate solutions, which is compatible with the large negative values of B22 measured. Conversely, lysozyme crystals were obtained under all conditions studied, and B22 values for this enzyme were within or close to the crystallization slot.

Importance of Protein Vibration Directionality on Function

Global protein vibrations have long been associated with protein functionality. Long-range intramolecular vibrations have been measured in proteins using anisotropic absorption in the terahertz frequency range [1,2]. These measurements directly correspond to the directionality of vibrations and show large changes in the directionality of the vibrational displacements for free chicken egg white lysozyme (CEWL) and inhibitor bound CEWL. Normal mode ensemble analysis (NMEA) and quasiharmonic analysis (QHA) calculations of the free and tri-acetylglucosamine (3NAG) bound CEWL dynamics were performed and also show large changes in vibration directionality with binding. This in spite of the calculated energy distribution showing very little change, in agreement with neutron scattering measurements. We investigate the importance of the protein directionality by extending the calculations to a double deletion mutant (DD CEWL) with an activity rate 1.4 times that of WT [3]. The deletions are far from the active site, indicating that the increased activity arises from changes in the dynamics. The calculated anisotropic spectra show large changes with mutation, whereas the energy distribution show practically no change from the WT. Similarities in the bound WT, free DD, and bound DD CEWL NMEA spectra indicate the mutation may be changing the directionality of the vibrations toward more efficient motions. Projections of the low frequency vibrations, from QHA, on the functional displacement show higher overlap of residues around Glu35 and Asp52 in the mutant system. These residues are known to be critical in CEWL functionality. The results reveal that the mutation may be steering the protein toward more functional dynamics without significantly affecting the energy distribution. [1] K.A. Niessen, et al. (2015) DOI: 10.1007/s12551-015-0168-4[2] G. Acbas, et al. (2014) DOI: 10.1038/ncomms4076[3] S. Mine, et al. (1999) DOI: 10.1006/jmbi.1999.2572. Supported by NSF (DBI 1556359, MCB 1616529) and DOE (DE-SC0016317).

Structural study of lysozyme in two ionic liquids at cryogenic temperature

We have investigated the structure and activity of chicken egg-white lysozyme in aqueous solutions of two typical ionic liquids, 1-butyl-3-methylimidazolium chloride and ethylammonium nitrate, at cryogenic temperature. An increase in structural disorder due to the unfolding and a decrease in the α-helical structure of lysozyme were noticeable upon glass formation. However, a decrease in the structural stability after cooling was less than that before cooling. The secondary and tertiary structures showed good reversibility upon cooling to 77K and then reverting back to ambient temperature. We discussed an influence of a cooling upon the structure in aqueous ionic liquid solutions.

Electrochemical Protein Cleavage in a Microfluidic Cell with Integrated Boron Doped Diamond Electrodes.

Specific electrochemical cleavage of peptide bonds at the C-terminal side of tyrosine and tryptophan generates peptides amenable to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification. To this end we developed a microfluidic electrochemical cell of 160 nL volume that combines a cell geometry optimized for a high electrochemical conversion efficiency (>95%) with an integrated boron doped diamond (BDD) working electrode offering a wide potential window in aqueous solution and reduced adsorption of peptides and proteins. Efficient cleavage of the proteins bovine insulin and chicken egg white lysozyme was observed at 4 out of 4 and 7 out of 9 of the predicted cleavage sites, respectively. Chicken egg white lysozyme was identified based on 5 electrochemically generated peptides using a proteomics database searching algorithm. These results show that electrochemical peptide bond cleavage in a microfluidic cell is a novel, fully instrumental approach toward protein analysis and eventually proteomics studies in conjunction with mass spectrometry.

Exploration of electrostatic interaction in the hydrophobic pocket of lysozyme: Importance of ligand-induced perturbation of the secondary structure on the mode of binding of exogenous ligand and possible consequences

Exogenous ligand binding can be adequate to alter the secondary structure of biomolecules besides other external stimuli. In such cases, structural alterations can complicate on the nature of interaction with the exogenous molecules. In order to accommodate the exogenous ligand, the biomolecule has to unfold resulting in a considerable change to its properties. If the bound ligand can be unbound, the biomolecule gets the opportunity to refold back and return to its native state. Keeping this in mind, we have purposely investigated the interaction of tartrazine (TZ), a well abundant azo food colorant, with two homologous lysozymes, namely, human lysozyme (HLZ) and chicken egg white lysozyme (CEWLZ) in physiological pH condition. The binding of TZ with lysozymes has been identified to accompany a ligand-induced secondary structure alteration as indicated by the circular dichroism spectra, and the reduction of α-helical content is more with HLZ than CEWLZ. Interestingly, the binding is identified to occur in the electronic ground state of TZ with lysozyme in its hydrophobic cavity, containing excess of positive charge, predominantly via electrostatic interaction. With increase of salinity of the medium the protein tends to refold back due to wakening of electrostatic forces and consequent reduction of strength of ligand interaction and unbinding. The entropy enthalpy compensation (EEC) has been probed to understand the binding features and it is found that CEWLZ-TZ shows better compensation than HLZ-TZ complex. This is presumably due to the fact that with CEWLZ the binding does not accompany substantial change in the protein secondary structure and hence ineffective to scramble the EEC. The present study initiates the importance of ligand-perturbed structural alteration of biomolecule in controlling the thermodynamics of binding. If there is a considerable alteration of the protein secondary structure due to binding, it is indicative that such changes should bring in the overall loss of activity of protein.

Optical Absorbance Sensitivity to Rugged Energy Landscape

Previously we have shown that underdamped long-range intramolecular vibrations exist in proteins using anisotropic absorption in the terahertz frequency range [1,2]. Measurements on free chicken egg white lysozyme (CEWL) and CEWL bound to the inhibitor tri-acetylglucosamine (3NAG) found the anisotropic absorption dramatically changes with inhibitor binding. To associate the spectral features with specific motions that are enhanced or diminished by the binding, we compare the measured spectra to calculations using normal mode analysis (NMA). Starting structures were randomly chosen frames from a molecular dynamics trajectory of a fully hydrated CEWL molecule. For each starting structure, we minimize the energy with small structural perturbations, and then perform NMA using CHARMM. These calculations find that the vibrational density of states (VDOS) is largely unchanged for a given starting structure, whereas the optical absorbance is different for each minimized structure. This variation arises from the sensitivity of the optical absorption to the strength and direction of the transition dipole, which is strongly dependent on the actual displacements for a given mode energy. This suggests that to properly model optical absorbance, one must perform an ensemble average of the calculated spectra, whereas for comparisons with the VDOS measurements, a single minimized structure is sufficient. The VDOS does not appear to be strongly affected by the ruggedness of the energy landscape, whereas the optical absorbance is. Given that strong bands are measured experimentally, the results suggest that in spite of the variation in the optical spectra, there are vibrations that are more prevalent than others, and possibly this biasing towards these motions enhances function. [1] Niessen, K.A., Xu, M., Markelz, A.G. (2015). Biophysical Reviews 7(2): 201-216. http://dx.doi.org/10.1007/s12551-015-0168-4. [2] Acbas, G., Niessen, K.A., Snell, E.H., Markelz, A.G. (2014) Nature Communications 5, 3076 http://dx.doi.org/10.1038/ncomms4076.

Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system.

ObjectivesThe effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic.ResultsBuffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme.ConclusionsPeptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s10529-015-2014-y) contains supplementary material, which is available to authorized users.

Molecular Design of Bisphosphonate-Modified Proteins for Efficient Bone Targeting In Vivo.

To establish a rational molecular design for bisphosphonate (BP)-modified proteins for efficient bone targeting, a pharmacokinetic study was performed using a series of alendronate (ALN), a nitrogen-containing BP, modified proteins with various molecular weights and varying degrees of modification. Four proteins with different molecular weight—yeast glutathione reductase (GR; MW: 112,000 Da), bovine serum albumin (BSA; MW: 67,000 Da), recombinant human superoxide dismutase (SOD; MW: 32,000 Da), and chicken egg white lysozyme (LZM; MW: 14,000 Da)—were modified with ALN to obtain ALN-modified proteins. Pharmacokinetic analysis of the tissue distribution of the ALN-modified and unmodified proteins was performed after radiolabeling them with indium-111 (111In) by using a bifunctional chelating agent. Calculation of tissue uptake clearances revealed that the bone uptake clearances of 111In-ALN-modified proteins were proportional to the degree of ALN modification. 111In-GR-ALN and BSA-ALN, the two high-molecular-weight proteins, efficiently accumulated in bones, regardless of the degree of ALN modification. Approximately 36 and 34% of the dose, respectively, was calculated to be delivered to the bones. In contrast, the maximum amounts taken up by bone were 18 and 13% of the dose for 111In-SOD-ALN(32) and LZM-ALN(9), respectively, because of their high renal clearance. 111In-SOD modified with both polyethylene glycol (PEG) and ALN (111In-PEG-SOD-ALN) was efficiently delivered to the bone. Approximately 36% of the dose was estimated to be delivered to the bones. In an experimental bone metastasis mouse model, treatment with PEG-SOD-ALN significantly reduced the number of tumor cells in the bone of the mice. These results indicate that the combination of PEG and ALN modification is a promising approach for efficient bone targeting of proteins with a high total-body clearance.

Extracellular expression of alkaline phytase in Pichia pastoris: Influence of signal peptides, promoters and growth medium

Alkaline phytase isolated from pollen grains of Lilium longiflorum (LlALP) possesses unique catalytic and thermal stability properties that suggest it has the potential to be used as a feed supplement. However, substantial amounts of active enzymes are needed for animal feed studies and endogenous levels of LlALP in lily pollen are too low to provide the required amounts. Active rLlALP2 (coded by LlAlp2, one of two isoforms of alkaline phytase cDNA identified in lily pollen) has been successfully expressed in intracellular compartments of Pichia pastoris, however enzyme yields have been modest (25–30mg/L) and purification of the enzyme has been challenging. Expression of foreign proteins to the extracellular medium of P. pastoris greatly simplifies protein purification because low levels of endogenous proteins are secreted by the yeast. In this paper, we first describe the generation of P. pastoris strains that will secrete rLlALP2 to the extracellular medium. Data presented here indicates that deletion of native signal peptides at the N- and C-termini of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion by four-fold; chicken egg white lysozyme signal peptide was ineffective in the extracellular secretion of rLlALP2. Second, we describe our efforts to increase expression levels by employing a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) in place of the strong, tightly controlled promoter of alcohol oxidase 1 gene (PAOX1). PGAP enhanced the extracellular expression levels of rLlALP2 compared to PAOX1. Finally, we report on the optimization of the culture medium to enhance yields of rLlALP2. The strength of PGAP varies depending on the carbon source available for cell growth; secreted expression of rLlALP2 was highest when glycerol was the carbon source. The addition of histidine and Triton X-100 also enhanced extracellular expression. Taken together, the employment of PGAP under optimized culture conditions resulted in approximately eight-fold (75–80mg/L) increase in extracellular activity compared to PAOXI (8–10mg/L). The P. pastoris expression system can be employed as a source of active alkaline phytase for animal feed studies.

Isolation and Characterization of Buffalo Milk Lysozyme

Lysozyme is a commercially valuable enzyme, and is applied in many fields, concerning products such as foods, drugs, and the like. In this work, lysozyme was isolated and purified from buffalo milk using sephadex G-50 and cation exchanger carboxymethyl cellulose. Lysozyme active fractions from buffalo milk were assayed against Gram positive substrate Micrococcus luteus at 450 nm and a decline in absorbance of 0.001 per min was observed. The optimum activity of lysozyme (158.3 ± 1.7 units/mL) was at 7.5 pH and 37°C temperature. Lysozyme activity at pasteurization temperatures 62.5°C, 30 min and 75°C, 15 s were (156.08 ± 1.03 and 156 ± 2 units/mL) not affected significantly; however, 47% activity of lysozyme was reduced at 100°C for 5 min. Antibacterial susceptibility testing of lysozyme (chicken egg white lysozyme and buffalo milk lysozyme) was performed on Micrococcus luteus (ATCC 4698) and Escherichia coli (ATCC 25235). Both lysozymes showed no inhibition effect against Escherichia coli.

Production and properties of tragacanthin-conjugated lysozyme as a new multifunctional biopolymer

In this communication we describe preparation and characterization of an enzyme-biopolymer conjugate composed of water-soluble part of gum tragacanth and chicken egg white lysozyme (LZM) under mild Maillard reaction conditions. SDS-PAGE together with FT-IR spectroscopy revealed that Maillard reactions occurred between LZM and tragacanthin (TRG). Under optimum conditions (pH = 8.5, 60 °C, RH = 79%, 8 days), approximately 2 TRG molecules were attached to one LZM molecule. DSC analysis showed that conjugation with TRG increased denaturation temperature by 6.35 °C. The resulting conjugates were characterized using scanning electron microscopy. The modified enzyme activity retained 77% of the original enzymatic activity after 8 days. TRG-conjugated LZM exhibited improved solubility and emulsion properties as compared with the native LZM. In addition, a significant increase in foam capacity and stability of LZM-TRG conjugate was detected. Conjugate with TRG significantly improved the inhibitory effect of LZM on the growth of Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella typhi in a dose dependent manner such that at 4000 μg/ml, LZM-TRG conjugate inhibited S. aureus, B. cereus, E. coli and S. typhi by 90%, 80%, 50% and 40%, respectively. Taken together these results suggest that the functional properties and antimicrobial activities of LZM can be improved by conjugation with TRG. The conjugation might expand the applications of LZM as a multifunctional ingredient in food and pharmaceutical industries.

Long-Range Protein Vibrations Dependence on Ligand Binding: Rate Promoting Motions

It has been suggested that long-range intramolecular vibrations in enzymes may provide efficient access to intermediate state configurations, enhancing catalytic turnover rates. Recently we have successfully measured these long-range protein vibrations using an anisotropic THz near-field technique to measure protein crystals, called crystal anisotropy terahertz microscopy (CATM). The method isolates these motions from the isotropic librational background, revealing narrow band resonances. Recent measurements on free chicken-egg white lysozyme (CEWL) and tri-N-acetylglucosamine (3NAG) inhibitor bound CEWL reveal dramatic and reproducible changes in the intramolecular vibrational mode spectra with binding. A large resonance at ∼70 cm−1 for free CEWL, is somewhat suppressed with binding, whereas a new resonance is observed at ∼ 40 cm−1, with binding. This dramatic change in the spectra with binding confirms that the observed resonances arise from intramolecular vibrations, and not crystal phonons. Using normal mode analysis, our calculated CATM spectra find a consistent increase in the low frequency signal with binding. While the density of states does not change appreciably with binding, the overall direction of motion shifts for vibrations in the frequency range where there is a large change in the optical signal. The change in the direction of the vibrational motion results in a change in the motion of the charge distribution and therefore, the dipole derivative, leading to the sensitivity to the trajectories in the terahertz optical absorbance. The remaining question is if these specific motions participate in the structural preorganization of the binding site, and thus promote catalytic activity. We will discuss measurements to answer this long standing question. This work was supported by NSF MRI∧2 grant DBI295998.

The Effect of Crystal Contact Forces on Protein Intramolecular Dynamics

Increasingly time resolved X-ray crystallography and solid state NMR have been employed to characterize dynamics. In the advent of X-ray free electron sources at Stanford (LCLS), and Hamburg (European XFEL) there is a strong push to extend time-resolved measurements. A persistent question for these techniques however, is how the crystal contact forces may strongly perturb these dynamics from those in vivo. While some theoretical studies have indicated that the crystal contact perturbation is minor[1], other calculations suggest it is significant[2]. Surprisingly there have been few studies to actually determine from the data what the effects are. Given the enormous effort currently underway for extending crystal phase dynamics measurements, it is imperative to determine how the crystal contact forces affect large scale motions necessary for function. Here we show how anisotropic optical measurements in the extreme infrared (10-100 cm-1) using the technique of Crystal Anisotropy Terahertz Microscopy (CATM) can quantify the effect [3], by measuring the perturbation of the global motions for a given symmetry group. Chicken egg white lysozyme (CEWL) is used as a benchmarking model. Calculations and measurements are performed for tetragonal and monoclinic symmetry groups, for which B-factor measurements indicate that there is a significant difference in the motional constraint arising from the crystal geometry.1. Hafner, J. and W.J. Zheng, Optimal modeling of atomic fluctuations in protein crystal structures for weak crystal contact interactions. Journal of Chemical Physics, 2010. 132(1).2. Hinsen, K., Analysis of domain motions by approximate normal mode calculations. Proteins: Structure, Function, and Genetics, 1998. 33(3): p. 417-429.3. Acbas, G., K.A. Niessen, E.H. Snell, and A.G. Markelz, Optical measurements of long-range protein vibrations. Nat Commun, 2014. 5.

Spatiotemporal development of soaked protein crystal.

Crystal soaking is widely performed in biological crystallography. This paper reports time-resolved X-ray crystallographic and microtomographic analyses of tetragonal crystals of chicken egg-white lysozyme soaked in mother liquor containing potassium hexachloroplatinate. The microtomographic analysis showed that X-ray attenuation spread from the superficial layer of the crystal and then to the crystal core. The crystallographic analyses indicated that platinum sites can be classified into two groups from the temporal development of the electron densities. A soaking process consisting of binding-rate-driven and equilibrium-driven layers is proposed to describe these results. This study suggests that the composition of chemical and structural species resulting from the soaking process varies depending on the position in the crystal.

Structural basis of protein oxidation resistance: a lysozyme study.

Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD) simulations, we find the protein parts with increased root-mean-square deviation (RMSD) to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation.

Modified lysozymes as novel broad spectrum natural antimicrobial agents in foods.

In recent years much attention and interest have been directed toward application of natural antimicrobial agents in foods. Some naturally occurring proteins such as lactoperoxidase, lactoferrin, and lysozyme have received considerable attention and are being considered as potential antimicrobial agents in foods. Lysozyme kills bacteria by hydrolyzing the peptidoglycan layer of the cell wall of certain bacterial species, hence its application as a natural antimicrobial agent has been suggested. However, limitations in the action of lysozyme against only Gram-positive bacteria have prompted scientists to extend the antimicrobial effects of lysozyme by several types of chemical modifications. During the last 2 decades extensive research has been directed toward modification of lysozyme in order to improve its antimicrobial properties. This review will report on the latest information available on lysozyme modifications and examine the applicability of the modified lysozymes in controlling growth of Gram-positive and Gram-negative bacteria in foods. The results of modifications of lysozyme using its conjugation with different small molecule, polysaccharides, as well as modifications using proteolytic enzymes will be reviewed. These types of modifications have not only increased the functional properties of lysozyme (such as solubility and heat stability) but also extended the antimicrobial activity of lysozyme. Many examples will be given to show that modification can decrease the count of Gram-negative bacteria in bacterial culture and in foods by as much as 5 log CFU/mL and in some cases essentially eliminated Escherichia coli. In conclusion this review demonstrates that modified lysozymes are excellent natural food preservatives, which can be used in food industry. The subject described in this review article can lead to the development of methods to produce new broad-spectrum natural antimicrobial agents, based on modification of chicken egg white lysozyme, which might potentially replace the currently used synthetic food preservatives.