The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

NMR-based localization of ions involved in salting out of hen egg white lysozyme

NaCl-induced aggregation of hen egg white lysozyme (HEWL) was monitored by NMR spectroscopy. Small, but significant, changes induced by salt addition in TOCSY spectra were attributed to the effect of local reorganization of protein backbone upon ion binding. Salt-induced variations in HN and H alpha chemical shifts were mapped on the HEWL 3D structure which allowed the construction of a scheme of the spatial localization of potential ion binding sites. It was found that in a 0.5 M NaCl solution six chloride anions and at least one sodium cation are bound to preferred sites on the HEWL surface.     

31P-NMR characterization of hen egg yolk and egg white

The avian egg is an isolated system where embryonic development can easily be studied. Furthermore, the system can be subjected to various environmental constraints. Its size is such that it can easily be accommodated in most NMR instruments. This is demonstrated by a recent 31P-NMR surface coil experiment where the increase of embryo size and development could be judged by the levels of ATP and phosphocreatine (Belton, P.S., Gordon, R.E., Jones, J.M. and Shaw, D. (1983) Br. Poult. Sci. 24, 429-433).     

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.     

Hydrolysis of whey by whole cells of Kluyveromyces bulgaricus immobilized in calcium alginate gels and in hen egg white

Summary Whey hydrolysis was compared in column reactors containing whole yeast cells immobilized in Ca-alginate or in hen egg white in relation to cell -galactosidase activity, flow rates, temperature and time. With cells of 1.3 U/mg dry weight (ONPG method) immobilized in Ca-alginate, 80% hydrolysis was obtained at 4° and 20° C with, respectively 0.50 and 1.65 bed volume/H; the values were 0.2 and 0.74 with cells entrapped in hen egg white. When the flow rate was expressed as ml/H/g wet yeast, no significant difference was observed between both matrices and 80% hydrolysis was reached with a flow rate 1.7 and 5 according to the temperature. The best performance was achieved by the yeast egg white reactor. At 4°C, hydrolysis decreased by 10% after 13 days; by 20% after 17 days. The presence of lactose transport inhibitors in whey did not significantly influence lactose hydrolysis.M. Decleire et al.: Hydrolysis of whey by immobilized whole cells of Kluyveromyces bulgaricus     

Viscoelastic properties of protein crystals: Triclinic crystals of hen egg white lysozyme in different conditions

A technique for the measurement of the dynamic Young's modulus E and logarithmic decrement ?? of protein crystals and other microscopic samples by the resonance method modified to a microscale is described. Monoclinic crystals of horse hemoglobin and sperm whale myoglobin; triclinic hen egg white lysozyme crystals, crosslinked by glutaraldehyde; and native and crosslinked needlelike lysozyme crystals were studied, as were amorphous protein films. The E of wet protein crystals is shown to be in the range (3–15) × 10³ kg/cm², ?? = 0.3–0.7. The crosslinking does not significantly affect the values. General elastic properties were analyzed for triclinic lysozyme crystals. No frequency dependence of E and ?? was found over the frequency range of 2.5–65 kHz. The temperature dependence was found to be characteristic for glassy polymers; the decrement of Young's modulus was ?2.4 ± 0.1%/°C. The guanidine HCl denaturation caused a 1000-fold decrease of E, its temperature dependence becoming similar to that of rubberlike materials. Studies of pH and salt effects showed E to be influenced by ionization of the acidic groups at pH 3–4.5. A humidity decrease from 100 to 30% caused a three- to fourfold increase of E and a decrease of ??. The final values of E = (40–60) × 10³ kg/cm² and ?? ? 0.1 were the same for dry crystals and amorphous films, whether crosslinked or not. These values may be attributed to the protein globular material.     

Structural details at active site of hen egg white lysozyme with di- and trivalent metal ions

Metal binding to lysozyme has received wide interest. In particular, it is interesting that Ni2+, Mn2+, Co2+, and Yb3+ chloride salts induce an increase in the solubility of the tetragonal form in crystals of hen egg white lysozyme at high salt concentration, but that Mg2+ and Ca2+ chloride salts do not. To investigate the interactions of the di- and trivalent metal ions with the active site of lysozyme and compare the effects of the di- and trivalent metal ions on molecular conformation of lysozyme based on the structural analysis, the crystal structures of hen egg white lysozyme grown at pH 4.6, in the presence of 0.5 M MgCl2, CaCl2, NiCl2, MnCl2, CoCl2, and YbCl3, have been determined by X-ray crystallography at 1.58 A resolution. The crystals grown in these salts have an identical space group, P4(3)2(1)2. The molecules show no conformational changes, irrespective of the salts used. Ni2+ and Co2+ binding to the Odelta atom of Asp52 in the active site at 1.98 and 2.02 A, respectively, and Yb3+ binding to both the Odelta atom of Asp52 and the Odelta1 atom of Asn46 at 2.25 A have been identified. The binding sites of Mn2+, Mg2+, and Ca2+ have not been found from different Fourier electron density maps. The Ni2+ and Co2+ ions bind to the Odelta atom of Asp52 at almost the same position, while the Yb3+ ion takes a different position from the Ni2+ and Co2+ ions. On the other hand, the anion Cl-, interacting with the Oeta atom of Tyr23 at a site of about 2.90 A, has also been determined for each crystal.     

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.     

Initial protein concentration and residual denaturant concentration strongly affect the batch refolding of hen egg white lysozyme

The effects of several variables on the refolding of hen egg white lysozyme have been studied. Lysozyme was denatured in both urea, and guanidine hydrochloride (GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1M Tris-HCl, pH 8.2, 1 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidised glutathione. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50°C. The apparent activation energy for lysozyme refolding was found to be 56 kJ/mol. Refolding by dilution results in low concentrations of both denaturant and reducing agent species. It was found that the residual concentrations obtained during dilution (100-fold dilution: [GuHCl]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1–10 mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT, higher refolding yields were obtained when starting from higher initial lysozyme concentrations. This trend was reversed when residual denaturant components were removed from the refolding buffer.     

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.     

Highly perturbed pKa values in the unfolded state of hen egg white lysozyme

The majority of pK_a values in protein unfolded states are close to the amino acid model pK_a values, thus reflecting the weak intramolecular interactions present in the unfolded ensemble of most proteins. We have carried out thermal denaturation measurements on the WT and eight mutants of HEWL from pH 1.5 to pH 11.0 to examine the unfolded state pK_a values and the pH dependence of protein stability for this enzyme. The availability of accurate pK_a values for the folded state of HEWL and separate measurements of mutant-induced effects on the folded state pK_a values, allows us to estimate the pK_a values of seven acidic residues in the unfolded state of HEWL. Asp-48 and Asp-66 display pK_a values of 2.9 and 3.1 in our analysis, thus representing the most depressed unfolded state pK_a values observed to date. We observe a strong correlation between the folded state pK_a values and the unfolded state pK_a values of HEWL, thus suggesting that the unfolded state of HEWL possesses a large degree of native state characteristics.     

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.     

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.     

Relation between hen egg white lysozyme and bacteriophage T4 lysozyme: Evolutionary implications

Hen egg white lysozyme and T4 bacteriophage lysozyme have the same catalytic function, but have non-homologous amino acid sequences. Notwithstanding the differences in their primary structures, the two lysozymes have similarities in their overall backbone conformations, in their modes of binding substrates and probably in their mechanisms of action.By different criteria, the similarity between the folding of the two enzymes can be shown to be statistically significant. Also the transformation which optimizes the agreement between the backbones of the two molecules is shown to accurately align their active site clefts, so that saccharide units bound in the A, B, C and D subsites of hen egg white lysozyme coincide within 1 to 2 Å with analogous saccharides bound to phage lysozyme. Furthermore, a number of the specific interactions between enzyme and substrate which were observed for hen egg white lysozyme, and thought to be important for catalysis, are found to occur in a structurally analogous way in the phage enzyme. Fifty-four atoms from the respective active sites which appear to be equivalent, including saccharides bound in the B and C sites, superimpose with a root-mean-square discrepancy of 1.35 Å.These structural and functional similarities suggest that the two lysozymes have arisen by divergent evolution from a common precursor. This is the first case in which two proteins of completely different amino acid sequence have been shown, with high probability, to have evolved by divergent rather than convergent evolution.