Re(CO)(3)(H(2)O)(3)(+) binding to lysozyme: structure and reactivity

The reaction of Re(CO)(3)(H(2)O)(3)(+) with hen egg white lysozyme in aqueous solution results in a single covalent adduct. Both NMR spectroscopy and single crystal X-ray diffraction show that the rhenium tricarbonyl cation binds to His15 via replacement of one of the coordinated water molecules. The formation of this adduct does not greatly affect the structure of the protein.     

Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model

The interactions of dodecyltrimethylammonium bromides (DTABs) with hen egg lysozyme have been investigated at pH = 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. DTAB interacts endothermically and activate lysozyme. The endothermicity of the lysozyme-DTAB interaction is in marked contrast to the exothermic interactions between sodium dodecyl sulphate (SDS) and lysozyme which have been attributed to specific binding between the anionic sulphate head groups and cationic amino acid residues. The enthalpies of interaction between the cationic surfactant (DTAB) and lysozyme are dominated by the endothermic unfolding of the native structure followed by an exothermic solvation of the lysozyme-DTAB complex by the addition of extra DTAB. A new direct calorimetric method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The extended solvation model was used to reproduce the enthalpies of lysozyme-DTAB interaction over the whole range of DTAB concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of DTAB, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The DTAB-induced denaturation enthalpy of lysozyme is 86.46 +/- 0.02 kJ mol(-1).     

Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe₃O₄. A thorough investigation of protein–MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe₃O₄, it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein–MNP interaction but also sheds light on the molecular mechanism underling this process.     

Production of bioactive lysophosphatidic acid by lysophospholipase D in hen egg white

Lysophosphatidic acid (LPA), a lysophospholipid mediator, is produced extracellularly by lysophospholipase D (lysoPLD) secreted in several animal body fluids including blood plasma. Previously, we reported that hen egg white contains polyunsaturated fatty acid-rich LPA. In this study, we examined whether lysoPLD is involved in the production of LPA in hen egg white. LysoPLD activity was measured by determining LPA and choline by mass spectrometric and enzyme-linked fluorometric analyses, respectively. LysoPLD increased with increased dilution of egg white, indicating that one or more components of egg white strongly inhibit its lysoPLD activity. This dilution-dependent increase in the lysoPLD activity was masked by co-incubation of the egg white with lysozyme, a major protein in hen egg white. Furthermore, addition of Zn(2+), Mn(2+), Ni(2+), or Co(2+) to diluted egg white altered preference patterns of lysoPLD toward choline-containing substrates. In particular, the egg white lysoPLD activity was greatly increased when Co(2+) was added. The cation-requirement of lysoPLD activity in hen egg white resembled that of plasma autotaxin (ATX)/lysoPLD. Western blot analysis revealed that egg white contained a protein that was immunostained with anti-ATX antibody. These results suggested that LPA in hen egg white is produced from lysophospholipids, especially LPC, by the action of ATX/lysoPLD, possibly originating from hen oviduct fluid.     

Comparison of the effects of 2,2,2-trifluoroethanol on peptide and protein structure and function

The co-solvent 2,2,2-trifluoroethanol (TFE) has been often used to aid formation of secondary structure in solution peptides or alternately as a denaturant within protein folding studies. Hen egg white lysozyme (HEWL) and a synthetic model peptide defining HEWL helix-4 were used as comparative model systems to systematically investigate the effect of increasing TFE concentrations on the structure of proteins and peptides. HEWL was analyzed using NMR, far-UV CD and fluorescence spectroscopy; with correlation of these results towards changes in enzymatic activity and the helix-4 peptide was analysed using NMR. Data illustrates two conflicting modes of interaction: Low TFE concentrations stabilize tertiary structure, observed from an increase in the number of NMR NOE contacts. Higher TFE concentrations denatured HEWL with the loss of lysozyme tertiary structure. The effects of TFE upon secondary structural elements within HEWL are distinct from those observed for the helix-4 peptide. This illustrates a dissimilar interaction of TFE towards both protein and peptide at equivalent TFE concentrations. The concentration that TFE promotes stabilization over denaturation is likely to be protein dependent although the structural action can be extrapolated to other protein systems with implications for the use of TFE in structural stability studies.     

Simulation of the thermal denaturation of hen egg white lysozyme: trapping the molten globule state.

In the study of protein folding, much attention has focused on the characterization of folding intermediates. We report here molecular dynamics simulations in which the initial stages of the thermal denaturation of hen egg white lysozyme in aqueous solution are examined in detail. It is found that lysozyme unfolds in a two-stage process with the initial formation a quasi-stable state in which significant rearrangement of the secondary structure takes place. No evidence for distinct folding domains was found. The simulations suggest that the formation of well-defined secondary structure occurs after the initial collapse of the peptide chain and thus tend against the framework model of protein folding.     

Three-dimensional structure of goose-type lysozyme from the egg white of the Australian black swan, Cygnus atratus

The egg white of C. atratus contains two forms of lysozyme, a 'chick-type' which is similar to that found in the egg white of the domestic hen, and a 'goose-type' similar to that found in the egg white of the Embden goose. The molecular structure of the goose-type lysozyme has been determined at a resolution of a 2.8 A by X-ray crystallographic analysis. The structure consists of two domains linked by a long stretch of alpha-helix. In all, there are seven helical segments in the structure. While there is no amino acid sequence homology with either hen egg-white or bacteriophage T4 lysozymes, there are portions of the structure where the folding of the main chain is similar to that found in portions of either hen egg-white lysozyme or T4 lysozyme or both. In particular, there is a consistency of structure in the arrangement of acid groups in the catalytic site. G-o plots calculated for this structure and for the bacteriophage T4 lysozyme structure show that both have similar 'modules' of structure with boundaries occurring at structurally equivalent positions. Three of the common boundaries are equivalent structurally to three of the four module boundaries observed in G-o plots of hen egg-white lysozyme. The variation in the position of the remaining boundary may be related to differences in substrate binding.     

Heterologous Expression of Hen Egg White Lysozyme and Resonance Assignment of Tryptophan Side Chains in its Non-native States

A new protocol is described for the isotope (¹⁵N and ¹³C,¹⁵N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. ¹H,¹³C and ¹⁵N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the C^γ resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.     

The influence of molecular variation on protein interactions

We investigated the influence of solvation forces on protein-protein interactions for two forms of lysozyme: hen egg white (HEWL) and turkey egg white (TEWL). Turkey egg white has more surface exposed hydrophobic residues than HEWL and the protein-protein interactions of TEWL are shown to be more attractive than those of HEWL, for the conditions studied. The importance of including a solvation term in the potential of mean force model, to account for molecular variation in protein surface characteristics, is highlighted. We also show that the magnitude of this solvation term can be estimated using readily available data.     

A stopped-flow fluorescence study of the native and modified lysozyme

The protein folding kinetics of hen egg white lysozyme (HEWL) was studied using experimental and bioinformatics tools. The structure of the transition state in the unfolding pathway of lysozyme was determined with stopped-flow kinetics using intact HEWL and its chemically modified derivative, in which six lysine residues have been modified. The overall consistency of φ-value (φ ≈ 1) indicates that lysine side chains interactions are subject to breaking in the structure of the transition state. Following experimental evidences, multiple sequence alignment of lysozyme family in vertebrates and exact structural examination of lysozyme, showed that the α-helix in the structure of lysozyme has critical role in the unfolding kinetics.     

Ionic liquids as refolding additives: N'-alkyl and N'-(omega-hydroxyalkyl) N-methylimidazolium chlorides

The purpose of this work was to investigate the influence of a series of N'-alkyl and N'-(omega-hydroxy-alkyl)-N-methylimidazolium chlorides on the renaturation of two model proteins, namely hen egg white lysozyme and the single-chain antibody fragment ScFvOx. All tested ionic liquids acted as refolding enhancers, with varying efficacies and efficiencies. The results of the refolding screening could be interpreted by taking into account the effect of the studied ionic liquids on protein aggregation, together with the systematic variations of their influence on the stability of native proteins in solution. More hydrophobic imidazolium cations carrying longer alkyl chains were increasingly destabilizing, while terminal hydroxylation of the alkyl chain made the salts more compatible with protein stability. The studied ionic liquids can be classified as preferentially bound, slightly to moderately chaotropic cosolvents for proteins.     

The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.     

Hydration dependence of conformational dielectric relaxation of lysozyme

Dielectric response of hen egg white lysozyme is measured in the far infrared (5-65 cm-1, 0.15-1.95 THz, 0.6-8.1 meV) as a function of hydration. The frequency range is associated with collective vibrational modes of protein tertiary structure. The observed frequency dependence of the absorbance is broad and glass-like. For the entire frequency range, there is a slight increase in both the absorbance and index of refraction with increasing hydration for <0.27 h (mass of H2O per unit mass protein). At 0.27 h, the absorbance and index begin to increase more rapidly. This transition corresponds to the point where the first hydration shell is filled. The abrupt increase in dielectric response cannot be fully accounted for by the additional contribution to the dielectric response due to bulk water, suggesting that the protein has not yet achieved its fully hydrated state. The broad, glass-like response suggests that at low hydrations, the low frequency conformational hen egg white lysozyme dynamics can be described by a dielectric relaxation model where the protein relaxes to different local minima in the conformational energy landscape. However, the low frequency complex permittivity does not allow for a pure relaxational mechanism. The data can best be modeled with a single low frequency resonance (nu approximately 120 GHz=4 cm-1) and a single Debye relaxation process (tau approximately .03-.04 ps). Terahertz dielectric response is currently being considered as a possible biosensing technique and the results demonstrate the required hydration control necessary for reliable biosensor applications.     

Raman optical activity characterization of native and molten globule states of equine lysozyme: comparison with hen lysozyme and bovine alpha-lactalbumin

Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine alpha-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22 degrees C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at approximately 1340 cm(-1), which is assigned to a hydrated form of alpha helix, is more similar to that in the ROA spectrum of bovine alpha-lactalbumin than hen lysozyme and may be associated with the greater flexibility and calcium-binding ability of equine lysozyme and bovine alpha-lactalbumin compared with hen lysozyme. In place of a strong sharp positive ROA band at approximately 1300 cm(-1) in hen lysozyme that is assigned to an alpha helix in a more hydrophobic environment, equine lysozyme shows a broader band centered at approximately 1305 cm(-1), which may reflect greater heterogeneity in some alpha-helical sequences. The ROA spectrum of apo equine lysozyme at pH 4.6 and 22 degrees C is almost identical to that of the holo protein, which indicates that loss of calcium has little influence on the backbone and side chain conformations, including the calcium-binding loop. From the similarity of their ROA spectra, the A state at pH 1.9 and both 2 and 22 degrees C and the apo form at pH 4.5 and 48 degrees C, which are partially folded denatured (molten globule or state A) forms of equine lysozyme, have similar structures that the ROA suggests contain much hydrated alpha helix. The A state of equine lysozyme is shown by these results to be more highly ordered than that of bovine alpha-lactalbumin, the ROA spectrum of which has more features characteristic of disordered states. A positive tryptophan ROA band at approximately 1551 cm(-1) in the native holo protein disappears in the A state, which is probably due to the presence of nonnative conformations of the tryptophans associated with a previously identified cluster of hydrophobic residues.     

Primary Structure of Lysozymes from Man and Goose

The primary structure of goose egg white lysozyme seems to be distinct from that of hen egg white and human leukaemia lysozymes.     

Protein structural perturbation and aggregation on homogeneous surfaces

We have demonstrated that globular proteins, such as hen egg lysozyme in phosphate buffered saline at room temperature, lose native structural stability and activity when adsorbed onto well-defined homogeneous solid surfaces. This structural loss is evident by alpha-helix to turns/random during the first 30 min and followed by a slow alpha-helix to beta-sheet transition. Increase in intramolecular and intermolecular beta-sheet content suggests conformational rearrangement and aggregation between different protein molecules, respectively. Amide I band attenuated total reflection/Fourier transformed infrared (ATR/FTIR) spectroscopy was used to quantify the secondary structure content of lysozyme adsorbed on six different self-assembled alkanethiol monolayer surfaces with -CH3, -OPh, -CF3, -CN, -OCH3, and -OH exposed functional end groups. Activity measurements of adsorbed lysozyme were in good agreement with the structural perturbations. Both surface chemistry (type of functional groups, wettability) and adsorbate concentration (i.e., lateral interactions) are responsible for the observed structural changes during adsorption. A kinetic model is proposed to describe secondary structural changes that occur in two dynamic phases. The results presented in this article demonstrate the utility of the ATR/FTIR spectroscopic technique for in situ characterization of protein secondary structures during adsorption on flat surfaces.     

Conformational features of reduced and disulfide intact forms of hen egg white lysozyme in aqueous solution in presence of 3-chloro-1, 2-propanediol and dioxane: implications for protein folding intermediates

Conformational features of reduced and disulfide intact hen egg white lysozyme in aqueous 1,4-dioxane and 3-chloro-1, 2-propanediol solutions have been examined using circular dichroism and fluorescence spectroscopy. We find that in presence of 1, 4-dioxane, reduced lysozyme assumes a relatively compact conformational form with secondary structure closer to native state and no tertiary structure as judged by peptide and aromatic CD spectra and ANS binding studies monitored by fluorescence. Further, in presence of 40% (v/v) 3-chloro-1, 2-propanediol, disulfide intact lysozyme (DI-lysozyme) assumes a conformational form with native like secondary structure and no tertiary structure akin to a molten globule state. We correlate our results to kinetic hydrogen- deuterium exchange NMR results of the refolding of lysozyme available in literature and suggest that the conformational forms observed in our study could be models for kinetic intermediates in the refolding of lysozyme.     

Structural study on hen egg-white lysozyme crystals grown in gravity and microgravity

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南极假丝酵母脂肪酶B在离子液体/超临界流体体系中的稳定性模拟

脂肪酶在离子液体/超临界流体体系中的结构稳定性是影响其活性的重要因素。本文采用分子动力学方法分别研究了南极假丝酵母脂肪酶B(CALB)在离子液体CYPHOS IL-201/极性超临界流体CHF_3两相体系和离子液体CYPHOS IL-201/非极性超临界流体CO_2两相体系中的结构稳定性,揭示影响CALB结构稳定性的因素。研究结果表明,在超临界CHF_3中,CHF_3破坏蛋白维持α螺旋结构的氢键是蛋白结构不稳定的主要原因;在超临界CO_2中,CALB蛋白的结构紧密性降低,有序二级结构发生了变化,导致稳定性下降。离子液体和两种超临界流体均形成了两相体系,蛋白处于离子液体相中,离子液体不溶于超临界流体,但超临界流体部分进入离子液体相,降低了离子液体相的黏度。其中,相比于CYPHOS IL-201/CO_2体系,CYPHOS IL-201/CHF_3体系的黏度降低多。在离子液体CYPHOS IL-201与超临界流体(CHF_3、CO_2)形成的两相体系中,离子液体CYPHOS IL-201具有保护蛋白结构的作用,使CALB蛋白结构更加稳定。     

Thermally induced fibrillar aggregation of hen egg white lysozyme

We study the effect of pH and temperature on fibril formation from hen egg white lysozyme. Fibril formation is promoted by low pH and temperatures close to the midpoint temperature for protein unfolding (detected using far-ultraviolet circular dichroism). At the optimal conditions for fibril formation (pH 2.0, T = 57 degrees C), on-line static light-scattering shows the formation of fibrils after a concentration-independent lag time of approximately 48 h. Nucleation presumably involves a change in the conformation of individual lysozyme molecules. Indeed, long-term circular dichroism measurements at pH 2.0, T = 57 degrees C show a marked change of the secondary structure of lysozyme molecules after approximately 48 h of heating. From atomic force microscopy we find that most of the fibrils have a thickness of approximately 4 nm. These fibrils have a coiled structure with a periodicity of approximately 30 nm and show characteristic defects after every four or five turns.