Organic cosolvents and hen egg white lysozyme folding

Studies on the influence of organic cosolvents on lysozyme folding have been reported. As most of the researches are confined to a few specific molecules and focus on equilibrium states, less is known about the effect on folding dynamics. We have studied the influence of six soluble organic cosolvents on hen egg white lysozyme heat induced denaturation and refolding dynamics. It was found that trifluoroethanol (TFE) can change the folding pathway significantly. With the presence of TFE, the overshot phenomenon generally observed in lysozyme folding at 222 nm disappears. The common mechanism of how organic cosolvents influence folding is analyzed. The heat induced denaturation temperature was found to have a quantitative relationship with the slow phase rate constant during folding. We discuss this finding and hypothesize that it is due to the similar influence of organic cosolvent on the transition state of heat denaturation and refolding.     

Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme

The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism.     

Effect of temperature on the interaction of cisplatin with the model protein hen egg white lysozyme

The products of the reaction between cisplatin (CDDP) and the model protein hen egg white lysozyme (HEWL) at 20, 37 and 55 °C in pure water were studied by UV–Vis absorption spectroscopy, intrinsic fluorescence and circular dichroism, dynamic and electrophoretic light scattering and inductively coupled plasma mass spectrometry. X-ray structures were also solved for the adducts formed at 20 and 55 °C. Data demonstrate that high temperature facilitates the formation of CDDP-HEWL adducts, where Pt atoms bind ND1 atom of His15 or NE2 atom of His15 and NH1 atom of Arg14. Our study suggests that high human body temperature (fever) could increase the rate of drug binding to proteins thus enhancing possible toxic side effects related to CDDP administration.     

Effect of ethanol on folding of hen egg-white lysozyme under acidic condition

The equilibrium and kinetics of folding of hen egg-white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride-induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two-state mechanism. The kinetics of denaturant-induced refolding and unfolding of hen egg-white lysozyme were investigated by stopped-flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far-UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near-UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on-pathway and off-pathway intermediates of protein folding.     

Dynamics of hydration in hen egg white lysozyme

We investigate the hydration dynamics of a small globular protein, hen egg-white lysozyme. Extensive simulations (two trajectories of 9 ns each) were carried out to identify the time-scales and mechanism of water attachment to this protein. The location of the surface and integral water molecules in lysozyme was also investigated. Three peculiar temporal scales of the hydration dynamics can be discerned: two among these, with sub-nanosecond mean residence time, tau(w), are characteristic of surface hydration water; the slower time-scale (tau(w) approximately 2/3 ns) is associated with buried water molecules in hydrophilic pores and in superficial clefts. The computed tau(w) values in the two independent runs fall in a similar range and are consistent with each other, thus adding extra weight to our result. The tau(w) of surface water obtained from the two independent trajectories is 20 and 24 ps. In both simulations only three water molecules are bound to lysozyme for the entire length of the trajectories, in agreement with nuclear magnetic relaxation dispersion estimates. Locations other than those identified in the protein crystal are found to be possible for these long-residing water molecules. The dynamics of the hydration water molecules observed in our simulations implies that each water molecule visits a multitude of residues during the lifetime of its bound with the protein. The number of residues seen by a single water molecule increases with the time-scale of its residence time and, on average, is equal to one only for the water molecules with shorter residence time. Thus, tau(w) values obtained from inelastic neutron scattering and based on jump-diffusion models are likely not to account for the contribution of water molecules with longer residence time.     

The stability curve of hen egg white lysozyme

The physiological stability curve--a plot of the free energy of unfolding versus temperature--is calculated for hen egg white lysozyme from a combination of extrapolated unfolding thermodynamic data from reversible conditions and isothermal titrations with guanidine hydrochloride. The shape of the curve suggests the existence of only one folded conformation.     

Stabilization of hen egg white lysozyme by a cavity-filling mutation

Stabilization of a protein using cavity-filling strategy has hardly been successful because of unfavorable van der Waals contacts. We succeeded in stabilizing lysozymes by cavity-filling mutations. The mutations were checked by a simple energy minimization in advance. It was shown clearly that the sum of free energy change caused by the hydrophobicity and the cavity size was correlated very well with protein stability. We also considered the aromatic-aromatic interaction. It is reconfirmed that the cavity-filling mutation in a hydrophobic core is a very useful method to stabilize a protein when the mutation candidate is selected carefully.     

Evidence for nucleation in the folding of reduced hen egg lysozyme

We have examined the early intermediate products of the regeneration of lysozyme by acidifying the regeneration solution containing the partially folded products, alkylating the free sulfhydryls and digesting the proteins sequentially with pepsin and chymotrypsin. Peptide maps were produced. Results indicate that a limited number of different disulfide bonds is formed early in the regeneration. From this we conclude that a limited number of 3-dimensional structures is formed in the early stages of the refolding process, and the process is not a random search.     

Molecular dynamics simulation of thionated hen egg white lysozyme

Understanding of the driving forces of protein folding is a complex challenge because different types of interactions play a varying role. To investigate the role of hydrogen bonding involving the backbone, the effect of thio substitutions in a protein, hen egg white lysozyme (HEWL), was investigated through molecular dynamics simulations of native as well as partly (only residues in loops) and fully thionated HEWL using the GROMOS 54A7 force field. The results of the three simulations show that the structural properties of fully thionated HEWL clearly differ from those of the native protein, while for partly thionated HEWL they only changed slightly compared with native HEWL. The analysis of the torsional-angle distributions and hydrogen bonds in the backbone suggests that the α-helical segments of native HEWL tend to show a propensity to convert to 3(10)-helical geometry in fully thionated HEWL. A comparison of the simulated quantities with experimental NMR data such as nuclear overhauser effect (NOE) atom-atom distance bounds and (3)J((H)(N)(H)(α))-couplings measured for native HEWL illustrates that the information content of these quantities with respect to the structural changes induced by thionation of the protein backbone is rather limited.     

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.     

Secretion of hen egg white lysozyme from Kluyveromyces lactis

Hen egg white (HEW) lysozyme was correctly processed and efficiently secreted from an alternative yeast, Kluyveromyces lactis. We constructed secretion vectors using PHO5, PGK, and LAC4 promoters, and found that the highest secretion was obtained under the direction of the PGK promoter in non-selective rich medium. K. lactis secreted HEW lysozyme with two-fold higher efficiency than S. cerevisiae, estimated by using a K. lactis-S. cerevisiae shuttle vector.     

Pressure effect on denaturant-induced unfolding of hen egg white lysozyme.

The influence of hydrostatic pressure (< or =100 MPa) on denaturant-induced unfolding of hen egg white lysozyme was investigated by means of ultraviolet spectroscopy at various temperatures. Assuming a two-state transition model, the dependence of Gibbs free-energy change of unfolding on the denaturant concentration was calculated. Under applied hydrostatic pressure, these data were interpreted as suggesting that a two-state model is not applicable in a restricted temperature range; the dominant effect of hydrostatic pressure is to affect the cooperativity in protein unfolding due to a chemical equilibrium shift in the direction of the reduction in the system volume. The deviation from the two-state transition model appears to be rationalized by assuming that applied pressure induces an intermediate conformation between the native and unfolded states of the protein. The implication of the thermodynamic stability of protein under pressure was discussed.     

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.     

Production of15N-labelled hen egg white lysozyme usingAspergillus niger

Summary Aspergillus niger has been used as a host organism for the production of¹⁵N-labelled hen egg white lysozyme (HEWL). In order to achieve maximum incorporation of label, strains expressing the HEWL gene were grown in medium containing ammonium¹⁵N chloride as sole nitrogen source. Yields of HEWL protein were reduced relative to those obtained on more complex media. Gains in yield using complex media were offset by reduction in¹⁵N incorporation. No differences in either yield or kinetics of production were observed when ammonium¹⁵N chloride was replaced by unlabelled ammonium chloride as sole nitrogen source. Yields of¹⁵N-HEWL produced in this way are adequate for, and offer considerable advantages to, NMR studies of structure and folding of mutant and wild-type lysozymes.     

The effect of tri-N-acetylglucosamine on hydrogen exchange in hen egg white lysozyme

Tritium-hydrogen isotope exchange techniques have been employed to study the effect of tri-N-acetylglucosamine binding on the conformational dynamics of hen egg white lysozyme. Numerical Laplace inversion of the data provides exchange rate probability density functions that reveal three overlapping peaks for both the free enzyme and (GlcNAc)3-enzyme complex. Binding of (GlcNAc)3 decreases the exchange rates of all protons to some extent with by far the largest effect being observed for the slow exchanging protons. These have been located, by comparison with neutron diffraction results (Mason, S. A., Bentley, G. A., and McIntyre, G. J. (1984) in Neutrons in Biology (Schoenborn, B. P., ed) pp. 323-334, Plenum Press, New York), within the beta-sheet structure and on helices (8-13), (28-34), and (89-97), that define the edges of the so-called "hydrophobic box" in lysozyme. The regions of the protein that are most affected by binding (GlcNAc)3, as revealed by hydrogen exchange, are found to be quite distinct from the regions observed to undergo conformational changes by x-ray diffraction. Most of these segments of the protein are located at some distance from the (GlcNAc)3-binding site itself. Two segments (the beta-sheet and helix (28-34)) are closely associated with the two active-site carboxylate groups. These results suggest that exchange-stable regions having strong, highly organized hydrogen bonding may have an important role in catalytic function and the differential propagation of conformational and dynamic perturbations caused by ligand binding at distant sites on the protein.     

Anti-inflammatory effect of lysozyme from hen egg white on mouse peritoneal macrophages

Lysozyme from hen egg has been reported to possess an anti-inflammatory effect. However, little is known about its detailed mechanism. The mechanism of anti-inflammatory effect of lysozyme was examined in this study. When mouse macrophage-like cell line RAW264.7 cells and mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and then treated with lysozyme, the production of tumor necrosis factor-α and interleukin-6 was significantly suppressed. The effect was induced by suppressing the gene expression levels of both cytokines. Phagocytosis activity of peritoneal macrophages was not altered by the treatment with lysozyme, suggesting that lysozyme shows the anti-inflammatory effect without inhibiting the phagocytotic response of macrophages. In addition, lysozyme inhibited phosphorylation of c-jun N-terminal kinase (JNK) and was taken up by macrophages within 1 h after treatment of the cells with lysozyme. Overall results suggest that lysozyme is taken up intracellularly and suppresses LPS-induced inflammatory responses by inhibiting JNK phosphorylation.     

L-Arginine increases the solubility of unfolded species of hen egg white lysozyme

L-Arginine (L-Arg) has been widely used as an enhancer of protein renaturation. The mechanism behind its action is still not fully understood. Using hen egg white lysozyme as a model protein, we present data that clearly demonstrate the suppression of the aggregation of denatured protein by L-Arg. By chemical modification of free cysteines, a series of unfolded lysozyme species were obtained that served as models for unfolded and intermediate states during the process of oxidative refolding. An increased equilibrium solubility of unfolded species and intermediates in the presence of L-Arg seems to be its major mechanism of action.     

1H-NMR study on the chitotrisaccharide binding to hen egg white lysozyme.

Interaction between hen egg white lysozyme and chitotrisaccharide was investigated by 1H-NMR spectroscopy using partially acetylated chitotrisaccharides and chemically modified lysozyme. Monoacetyl (GlcN-GlcN-GlcNAc), diacetyl (GlcN-GlcNAc-GlcNAc), or triacetyl chitotrisaccharide [(GlcNAc)3] was added to the lysozyme solution, and the changes in the 1H-NMR signals of the lysozyme were analyzed. Although many of the resonances were affected by addition of the saccharide, the most remarkable effect was seen on the signal of Trp28 C5H which is in a hydrophobic box adjacent to the saccharide-binding site. The signal shifted upfield by 0.2 ppm upon (GlcNAc)3 binding, whereas the chemical shift change of the signal resulting from binding of GlcN-GlcNAc-GlcNAc or GlcN-GlcN-GlcNAc was smaller than that resulting from (GlcNAc)3 binding. When the Asp101-modified lysozyme was used instead of the native lysozyme, the chemical shift change of the Trp28 C5H signal resulting from (GlcNAc)3 binding was also smaller than that for the native lysozyme. The chemical shift change of the signal reflects the conformational change of the hydrophobic box region which should synchronize with the movement of the binding site resulting from the saccharide binding. Therefore, the conformational change resulting from the saccharide binding might be reduced when the sugar residues located at binding subsites A and B of the lysozyme are deacetylated, as well as when Asp101 interacting with the sugar residues at the same subsites is modified.