Turkey Egg-white Lysozyme Research Articles

Crystal growth of lysozymes in media contaminated by parent molecules: influence of gelled media

A series of solution and gel growth experiments have been done to obtain hen egg white lysozyme (HEWL) crystals in media contaminated by a parent molecule, turkey egg white lysozyme (TEWL) or TEWL crystals from media contaminated by HEWL. In both cases, such a contamination causes nucleation inhibition and morphology changes. Evolutions of nucleation and morphology as a function of contaminant percentage show that the contaminant percentage is efficient on nucleation at low percentages (< 10%) and on morphology at high percentage (> 20%). This suggests that the contaminant exists in solution as different species probably oligomers, whose action is different for prenucleation and growth processes. Gel media extend the domain for which usable monocrystals are obtained from 50% contaminant (case of solution) to 60% (case of agarose gel) or 70% (case of silica gel).

Structure of hexagonal turkey egg-white lysozyme at 1.65A resolution.

The structure of hexagonal turkey egg-white lysozyme (TEWL) has been determined and refined at 1.65 A resolution. The crystals were grown from a 150 mM potassium thiocyanate solution at pH 4.5 and belong to space group P6(1)22 with unit-cell dimensions a = b = 70.96, c = 83.01 A alpha = beta = 90, gamma = 120 degrees. The crystals were isomorphous with those of hexagonal pH 8.0 TEWL. The coordinates of PDB entry code 3LZ2 were therefore used as the initial model and subjected to rigid-body refinement, simulated annealing and least-squares refinement to a final residual of 0.20. The root-mean-square deviations from the ideal bond distances and angles were 0.016 A and 2.2 degrees, respectively. During the refinement, 86 water molecules and one thiocyanate ion were located in the structure. The thiocyanate ion lies close to the interface between two symmetry-related molecules. The S atom of the ion forms two direct intermolecular contacts with Argl4 and interacts indirectly via a network of water molecules to Arg5 of a symmetry-related molecule. The structure provides direct evidence for the mode of thiocyanate binding to arginine residues and suggests a possible mechanism for the efficiency of thiocyanate in crystallizing basic proteins.

Measurement of the individual pKa values of acidic residues of hen and turkey lysozymes by two-dimensional 1H NMR

The pH dependence of the two-dimensional 1H nuclear magnetic resonance spectra of hen and turkey egg-white lysozymes has been recorded over the pH range 1-7. By monitoring the chemical shifts of the resonances of the various protons of ionizable residues, individual pKa values for the acidic residues have been determined for both proteins. The pKa values are displaced, with the exception of those of the residues in the active site cleft, by an average of 1 unit to low pH compared to model compounds.

By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.

By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection. Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage. Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues. The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA). Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range. The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3. However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin. Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.

Structure determination of turkey egg-white lysozyme using Laue diffraction data

The three-dimensional structure of turkey egg-white lysozyme (TEWL) has been solved and refined at 2.5 A resolution using X-ray data collected by the Laue method. This is the first protein structure determination undertaken using Laue diffraction data. A re-examination of the existing structure of TEWL was necessary when attempts to refine an atomic model based on the C alpha positions in the Protein Data Bank (entry 1LZ2) failed. The correct orientation and position of the turkey lysozyme molecules within the crystallographic unit cell were determined by molecular replacement using a refined model of the homologous hen egg-white lysozyme crystal structure. After modification of the model to reflect the differences in amino-acid sequence between the chicken and turkey enzymes, the structure was subjected to crystallographic refinement using the simulated-annealing refinement technique and conventional least-squares refinement. This yielded a final residual of R = 20.7%. This crystal form is of potential interest for time-resolved crystallographic studies since the amino-acid residues involved in catalysis (Asp52 and Glu35) are accessible to solvent and not blocked by crystal contacts.

Activity of crystalline turkey egg white lysozyme.

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.

By-passing immunization: Human antibodies from V-gene libraries displayed on phage

We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (V H) and light (V κ and V λ chain variable (V) genes were prepared from peripheral blood lymphocytes (PBLs) of unimmunized donors by polymerase chain reaction (PCR) amplification. Genes encoding single chain Fv fragments were made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library (>10 7 members) cloned for display on the surface of a phage. Rare phage with “antigenbinding” activities were selected by four rounds of growth and panning with “antigen” (turkey egg-white lysozyme (TEL) or bovine serum albumin) or “hapten” (2-phenyloxazol-5-one (phOx)), and the encoding heavy and light chain genes were sequenced. The V-genes were human with some nearly identical to known germ-line V-genes, while others were more heavily mutated. Soluble antibody fragments were prepared and shown to bind specifically to antigen or hapten and with good affinities, K a (TEL) = 10 7 m −1; K a (phOx) = 2 × 10 6 m −1. Isolation of higher-affinity fragments may require the use of larger primary libraries or the construction of secondary libraries from the binders. Nevertheless, our results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.

A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.

The effect of protein contaminants on the crystallization of turkey egg white lysozyme

We report here a series of studies on the controlled contamination of crystallizing solutions of the hexagonal form of turkey egg white lysozyme (TEWL) carried out to understand the effects of impurities on the nucleation and growth of protein crystals. The contamination of TEWL solutions with any of three other avian lysozymes affects both the nucleation and the growth processes. For hen and quail egg white lysozymes, low and medium levels of contamination result in partial inhibition of nucleation and shortening of the c-axis. Further increase of the contaminant concentration leads to detectable co-crystallization. A different effect is obtained when using the pheasant egg white lysozyme. Contamination by an unrelated protein, ribonuclease A, has an effect on the nucleation levels that is similar to those observed with the avian lysozymes. However, no effect on TEWL crystal morphology is observed. Thus, in the case of TEWL crystals, one can distinguish between a specific effect on crystal morphology induced by related proteins and a more general inhibitory effect on the nucleation levels observed in all cases studied here.

Overload Quasi-Static Linear Gradient Chromatography: Theory Versus Hydroxyapatite High-Performance Liquid Chromatography

Abstract The earlier theory of quasi-static linear gradient chromatography under overload conditions is improved. The quasi-crystalline phase model for the molecules adsorbed on the adsorbent surfaces in the column is specified; this model was introduced earlier, and it is used in the present theory. A close experimental verification of the theory is carried out for the case of hydroxyapatite high-performance liquid chromatography using both turkey and chicken egg white lysozymes as probes. It can be deduced that a quasi-crystalline phase of lysozyme molecules, in fact, is realized on the hydroxyapatite surfaces in the column. The mutual interaction energy per molecule of lysozyme occurring provided the maximum possible contact with other molecules is made on the hydroxyapatite surface can be estimated to be larger than 0.2–0.4 kcal/mol.

Crystallographic study of turkey egg-white lysozyme and its complex with a disaccharide

The crystal structure of turkey egg-white lysozyme, determined by the molecular replacement method at 5 Å resolution (Bott & Sarma, 1976) has now been refined to 2.8 Å resolution and a model has been built to fit the electron density. A comparison of the co-ordinates with those of hen lysozyme indicate a rootmean-square deviation of 1.6 Å for all the main-chain and side-chain atoms. A significant difference is observed in the region of residues 98 to 115 of the structure. The molecules are packed in this crystal form with the entire length of the active cleft positioned in the vicinity of the crystallographic 6-fold axis and is not blocked by neighboring molecules. A difference electron density map calculated between crystals of turkey lysozyme soaked in a disaccharide of N-acetyl glucosamine— N-acetyl muramic acid and the native crystals showed a strong positive peak at subsite C, a weak positive peak at subsite D and two strong peaks that correspond to the subsite E and a new subsite F′. This new site F′ is different from the subsite F predicted for the sixth saccharide from model building in hen lysozyme. The interactions between the saccharides bound at subsites E and F′ and the enzyme molecules are discussed.

Crystal structure of Turkey egg-white lysozyme: Results of the molecular replacement method at 5 Å resolution

Turkey egg-white lysozyme differs from hen egg-white lysozyme in its primary structure in 7 of the 129 residues. We have determined the rotational and translational parameters relating the known co-ordinates of hen egg-white lysozyme molecule to the turkey lysozyme. The rotational parameters were determined using the rotation function, the translational parameters were determined by placing the properly rotated molecule systematically at all positions within the unit cell and searching for those positions producing few intermolecular contacts between the α-carbon atoms of one molecule and all its neighbors. These parameters were refined by minimizing the conventional R factor between observed and calculated structure amplitudes. The final rotational and translational parameters give an R value of 46.7% for reflections with d spacings between 6 Å and 12 Å and have 7 intermolecular contacts closer than 5 Å between the a carbon atoms of one molecule and all its neighbors. An electron density map has been calculated at 5 Å resolution; the packing of the molecules in this form appears to present the entire length of the active cleft in the vicinity of the crystallographic 6-fold axis and does not appear to be blocked by neighboring molecules.

Optical properties of lysozyme. pH and saccharide binding difference spectra.

Difference spectra associated with changes in pH and with binding of saccharides have been recorded for hen egg white (HEW) lysozyme, turkey egg white (TEW) lysozyme, and for the derivatives of the hen protein in which Tre-62 or Trp-108 had been oxidized specifically to oxindolealanine to give the Oxa-62 or Oxa-108-proteins. Identical pH difference spectra were obtained for HEW, TEW, and Oxa-62-lysozymes. Oxidation of Trp-108 is reflected in both the high and low pH (pH 7 versus 5 and pH 2 versus 5) difference spectra. The magnitude of the low pH difference spectrum is enhanced by binding of saccharide for HEW and Oxa-62-lysozymes but not for TEW lysozyme. The shapes and magnitudes of saccharide binding difference spectra are affected by oxidation of residues 62 or 108. These results can be interpreted in terms of the perturbations responsible for the lysozyme difference spectra. The pH 7 versus 5 difference spectrum results from perturbation by Glu-35 of Trp-108 and another tryptophan, probably Trp-63. Perturbation of Trp-108 and one or more other tryptophan residues by several carboxylate groups is responsible for the low pH difference spectra of the unliganded HEW and TEW lysozyme molecules. Perturbation of Trp-108 makes a principal contribution to the saccharide-binding difference spectrum. Perturbation of the Oxa-108 chromophore by ionization of Glu-35 or by saccharide binding produces absorbance changes in the 250 to 265 nm region.

Turkey egg white lysozyme. Free energy, enthalpy, and steady state kinetics of reaction with N-acetylglucosamine oligosaccharides.

Equilibrium and calorimetric studies of substrate binding to turkey egg white (TEW) lysozyme were carried out at 30degrees as a function of pH (2 to 9) and ligand size (monosaccharide to hexasaccharide of N-acetylglucosamine). Steady state kinetic measurements using the N-acetylglucosamine hexasaccharide were carried out as a function of pH (2 to 9) and temperature (20-60degrees). These experiments allow comparison of the properties of TEW lysozyme with those of the hen egg white (HEW) enzyme reported previously (Banerjee, S. K., Holler, E., Hess, G. P., and Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367, and references therein). The free energies and enthalpies of oligosaccharide binding are the same for TEW and HEW lysozymes at pH 2 but are less negative for TEW lysozyme at pH 5. The pH dependence of the binding of (GlcNAc)3 and higher oligomers to TEW lysozyme is like that for the binding of beta-methyl-N-acetylglucosaminide to TEW lysozyme. These data indicate that oligosaccharide ligands bind identically with HEW and TEW lysozymes, except for the interactions of residue 101, which is aspartic acid in the HEW protein and glycine in the TEW protein (Larue, J. N., and Speck, J. C., Jr. (1970) J. Biol. Chem. 245, 1985-1991). The pH dependence of kcat is described by apparent pK values of 3.9 and 6.8 and a maximum value of kcat of 0.135 s-1. A value of 21.0 kcal/mol was calculated for deltaH from the temperature dependence of kcat. These values and the dependence of the transglycosylation reaction on acceptor concentration are within experimental error the same as those for HEW lysozyme. The more acid pK seen in the pH rate profile reflects the ionization of Asp-52 in the lysozyme-(GlcNAc)6 complex. The pK of Asp-52 in the free protein is 0.3 pK unit lower. The essential identity of the active sites of the HEW and TEW enzymes, except for the Asp-101 interactions, allows estimation of the thermodynamic properties associated with formation of the two hydrogen bonds between Asp-101 and substrate as deltaG0 = -1.2 kcal/mol, DeltaH0 = -3.6 kcal/mol, and deltaS0 = -7.9 e.u.

Participation of the Catalytic Carboxyls, Asp 52 and Glu 35, and Asp 101 in the Binding of Substrate Analogues to Hen Lysozyme1

The interactions of the substrate analogues, GlcNAc, beta-methyl GlcNAc, (GlcNAc)2, and (GlcNAc)3, with turkey egg-white lysozyme [ED 3.2.1.17], in which the Asp 101 of hen lysozyme is replaced by Gly, were studied at various pH values by measuring changes in the circular dichroic (CD) band at 295 nm. Results were compared with those for hen egg-white lysozyme. The modes of binding of these substrate analogues to turkey lysozyme were very similar to those hen lysozyme except for the participation of Asp 101 in hen lysozyme. The ionization constants of the catalytic carboxyls, Glu 35 and Asp 52, in the turkey lysozyme-(GlcNAc)3 complex were determined by measuring the pH dependence of the CD band at 304 nm, which originates from Trp 108 near the catalytic carboxyls. The ionization behavior of the catalytic carboxyls of turkey lysozyme in the presence and absence of (GlcNAc)3 was essentially the same as that for hen lysozyme. The pH dependence of the binding constant of (GlcNAc)3 to hen lysozyme was compared with that to turkey lysozyme between pH 2 and 8. The pH dependence of the binding constant for (GlcNAc)3 to turkey lysozyme could be interpreted entirely in terms of perturbation of catalytic carboxyls. In the case of hen lysozyme, it was interpreted in terms of perturbation of the catalytic carboxyls and Asp 101 in the substrate-binding site. The pK values of Asp 101 in hen lysozyme and the hen lysozyme-(GLcNAc)3 complex were 4.5 and 3.4, respectively. The binding constants of (GlcNAc)3 to lysozyme molecules with different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated. The binding constant of lysozyme, in which Asp 52 and Glu 35 are deprotonated, to (GlcNAc)3 was the smallest. The other three species had similar binding constant to (GlcNAc)3.

Nuclear magnetic resonance and ultraviolet difference spectral studies of the binding properties of turkey egg white lysozyme: Consequences of the replacement of ASP 101 by glycine

The nuclear magnetic resonance spectrum of the 19F nuclei in N-trifluoroacetylated chitotriose was studied in the presence of turkey lysozyme. In contrast to results previously obtained with hen lysozyme, the 19F nmr spectrum of the complex did not show any striking pH dependence. It was, in fact, very similar at all pH's to the spectrum of the trisaccharide complexed with hen lysozyme at low pH, where Asp 101 is protonated. The replacement of Asp 101 in turkey lysozyme by a glycine is thought to account for this difference and the results allow unequivocal assignment of a value of 4.2 to the pK a of Asp 101 in hen lysozyme. The dissociation constant of the chitotriose-turkey lysozyme complex was measured at various pH's using uv difference methods and compared with that previously reported for the hen lysozyme-chitotriose complex. Again, the results could be attributed to the loss in binding energy due to the absence of Asp 101. In contrast to chitotriose, the binding of chitobiose and methyl-2-acetamido-2-deoxy-β- d-glucopyranoside as studied by both uv difference and nmr methods is the same within experimental error for turkey and hen lysozyme. The results obtained for binding of chitobiose suggest that Asp 101 does not contribute as much to the binding energy of the disaccharide as was previously thought. Finally, the specific activities of both of these lysozymes against Micrococcus lysodeikticus were found to be identical.

Reaction of turkey egg-white lysozyme with tetranitromethane. Modification of tyrosine and tryptophan.

In a study of the reaction of tetranitromethane with turkey egg-white lysozyme at pH 8.5, it has been found that the number of 3-nitrotyrosine residues formed is dependent on the molar excess of tetranitromethane: 0.6 tyrosines nitrated with a 2.5 M excess; 1.3 with a 25 M excess; and 2.3 with a 250 M excess. Approximately one tyrosine remains unreactive, even with a 250 M excess of tetranitromethane. In each case, the product contains appreciable enzymatic activity: 100% for the 2.5:1 reaction; 48% for the 25:1 reaction; and 40% for the 250:1 reaction. Detectable polymerization of the lysozyme accompanies nitration, as indicated by gel filtration. In addition, the higher ratios of tetranitromethane to lysozyme lead to the modification of tryptophan as well as tyrosine. The most rapidly nitrated tyrosine is tyr-23 in the known sequence of the enzyme.Hydrolysis of the modified lysozymes with 3 N p-toluenesulfonic acid, under conditions normally used to determine the tryptophan content, leads to total destruction of 3-nitrotyrosine.

Reversible inactivation of avian lysozymes by dimethyl (2-hydroxy-5-nitrobenzyl)-sulfonium bromide

The reaction of hen egg white lysozyme with a 4 molar excess of dimethyl (2-hydroxy-5-nitrobenzyl)-sulfonium bromide at pH 6.0 leads to total loss of enzymatic activity within 5 minutes. Upon standing, the inactivated enzyme spontaneously regains activity, leveling off at 60% of the original activity after 72 hours. Under the same conditions, turkey egg white lysozyme is reduced to less than 5% of its original activity within 5 minutes, then spontaneously reactivates to 85% of its original activity after 24 hours. Human lysozyme shows no dramatic loss of activity when treated under these conditions. The presence of the substrate, chitotetraose, prevents the initial inactivation of both hen and turkey enzymes.

Cross-reactivity of some rabbit anti-human lysozyme sera with gallinaceous lysozymes

Sera from several rabbits immunized with human lysozyme were found to agglutinate hen egg-white lysozyme-coated sheep red blood cells. When assayed for direct binding with 125I-labeled hen egg-white lysozyme, these sera were found to give approximately 5–10% of the binding found with 125I-labeled human lysozyme. This binding was inhibitable by unlabelled human lysozyme. Binding similar to that of hen egg-white lysozyme was found with Japanese quail lysozyme. However, 125I-labeled turkey egg-white lysozyme was found to be bound to a greater extent. An explanation offered for the greater cross-reactivity of turkey lysozyme with anti-human lysozyme, as compared to that found with hen or Japanese quail lysozyme, invokes the presence of Leu instead of His at position 15 in turkey and human lysozymes.

Turkey Egg White Lysozyme: PREPARATION OF THE CRYSTALLINE ENZYME AND INVESTIGATION OF THE AMINO ACID SEQUENCE

Crystalline turkey egg white lysozyme has been prepared by a simple procedure consisting of adsorption on carboxymethylcellulose followed by elution with (NH4)2CO3 solution and crystallization from 5% NaCl solution. The twice crystallized enzyme gave a single peak when eluted from carboxymethylcellulose, and it migrated as a single band during disc electrophoresis. Separation of the tryptic peptides from reduced and S-carboxymethylated turkey egg white lysozyme has yielded peptides analogous to those of reduced and S-carboxymethylated chicken egg white lysozyme. It is found that the turkey enzyme amino acid sequence differs from that of the chicken lysozyme at a minimum of seven positions which are distributed over six of these tryptic peptides. The replacements detected from examination of these peptides are: Tyr for Phe3, Leu for His15, His for Gln41, Lys for Arg73, Ala for Val99, Gly for Asp101, and His for Gln121 of the chicken egg white lysozyme. The apparent occurrence of glycine as Residue 101 of turkey egg white lysozyme is especially interesting inasmuch as the x-ray crystallographic investigation of chicken egg white lysozyme and its complexes with certain inhibitors has led to the view that the carboxyl group of Asp101 is involved in binding substrates.