Contaminant inclusion into protein crystals analyzed by electrospray mass spectrometry and X-ray crystallography.

The inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X-ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X-ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results.     

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.     

Crystal structure of a phage library-derived single-chain Fv fragment complexed with turkey egg-white lysozyme at 2.0 A resolution

The three-dimensional structure of the single-chain Fv fragment 1F9 in complex with turkey egg-white lysozyme (TEL) has been determined to a nominal resolution of 2.0 A by X-ray diffraction. The scFv fragment 1F9 was derived from phage-display libraries in two steps and binds both hen and turkey egg-white lysozyme, although the level of binding affinity is two orders of magnitude greater for the turkey lysozyme. The comparison of the crystal structure with a model of the single-chain Fv fragment 1F9 in complex with hen egg-white lysozyme (HEL) reveals that in the latter a clash between Asp101 in lysozyme and Trp98 of the complementarity determining region H3 of the heavy chain variable domain occurs. This is the only explanation apparent from the crystal structure for the better binding of TEL compared to HEL.The binding site topology on the paratope is not simply a planar surface as is usually found in antibody-protein interfaces, but includes a cleft between the light chain variable domain and heavy chain variable domain large enough to accommodate a loop from the lysozyme. The scFv fragment 1F9 recognizes an epitope on TEL that differs from the three antigenic determinants recognized in other known crystal structures of monoclonal antibodies in complex with lysozyme.     

The occurrence of uridine diphosphate N-acetylgalactosamine 6-sulfate in quail egg white and characteristic distribution of sulfated sugar nucleotides in different avian eggs

A sulfated sugar nucleotide has been isolated from quail egg white, and accounts for nearly 80% of the total sugar nucleotides found in the egg white. Evidence is presented that this nucleotide is uridine diphosphate N-acetylgalactosamine 6-sulfate, an isomer of the 4-sulfated derivative of uridine diphosphate N-acetylgalactosamine previously found in chicken egg white. Further studies on the distribution of sulfated sugar nucleotides in egg white of various birds (chicken, quail, pheasant, peafowl, turkey, goose, and duck) demonstrate that each species has a characteristic composition, differing from one another regarding the relative amounts of 4-sulfated, 6-sulfated, and 4,6-bissulfated derivatives of uridine diphosphate N-acetylgalactosamine.     

Crystallographic determination of the mode of binding of oligosaccharides to T4 bacteriophage lysozyme: Implications for the mechanism of catalysis

Phage lysozyme has catalytic activity similar to that of hen egg white lysozyme, but the amino acid sequences of the two enzymes are completely different.The binding to phage lysozyme of several saccharides including N-acetylglucosamine (GlcNAc), N-acetylmuramic acid (MurNAc) and (GlcNAc)₃ have been determined crystallographically and shown to occupy the pronounced active site cleft. GlcNAc binds at a single location analogous to the C site of hen egg white lysozyme. MurNAc binds at the same site. (GlcNAc)₃ clearly occupies sites B and C, but the binding in site A is ill-defined.Model building suggests that, with the enzyme in the conformation seen in the crystal structure, a saccharide in the normal chair configuration cannot be placed in site D without incurring unacceptable steric interference between sugar and protein. However, as with hen egg white lysozyme, the bad contacts can be avoided by assuming the saccharide to be in the sofa conformation. Also Asp20 in T4 lysozyme is located 3 Å from carbon C₍₁₎ of saccharide D, and is in a position to stabilize the developing positive charge on a carbonium ion intermediate. Prior genetic evidence had indicated that Asp20 is critically important for catalysis. This suggests that in phage lysozyme catalysis is promoted by a combination of steric and electronic effects, acting in concert, The enzyme shape favors the binding in site D of a saccharide with the geometry of the transition state, while Asp20 stabilizes the positive charge on the oxocarbonium ion of this intermediate. Tn phage lysozyme, the identity of the proton donor is uncertain. In contrast to hen egg white lysozyme, where Glu35 is 3 Å from the glycosidic DOE bond, and is in a non-polar environment, phage lysozyme has an ion pair, Glull … Arg145, 5 Å away from the glycosidic oxygen. Possibly Glull undergoes a conformational adjustment in the presence of bound substrate, and acts as the proton donor. Alternatively, the proton might come from a bound water molecule.     

Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes.

On the basis of the molecular evolution of hen egg white, human, and turkey lysozymes, three replacements (Trp62 with Tyr, Asn37 with Gly, and Asp101 with Gly) were introduced into the active-site cleft of hen egg white lysozyme by site-directed mutagenesis. The replacement of Trp62 with Tyr led to enhanced bacteriolytic activity at pH 6.2 and a lower binding constant for chitotriose. The fluorescence spectral properties of this mutant hen egg white lysozyme were found to be similar to those of human lysozyme, which contains Tyr at position 62. The replacement of Asn37 with Gly had little effect on the enzymatic activity and binding constant for chitotriose. However, the combination of Asn37----Gly (N37G) replacement with Asp101----Gly (D101G) and Trp62----Tyr (W62Y) conversions enhanced bacteriolytic activity much more than each single mutation and restored hydrolytic activity toward glycol chitin. Consequently, the mutant lysozyme containing triple replacements (N37G, W62Y, and D101G) showed about 3-fold higher bacteriolytic activity than the wild-type hen lysozyme at pH 6.2, which is close to the optimum pH of the wild-type enzyme.     

Re-evaluation of intramolecular long-range electron transfer between tyrosine and tryptophan in lysozymes. Evidence for the participation of other residues.

One-electron oxidation of six different c-type lysozymes from hen egg white, turkey egg white, human milk, horse milk, camel stomach and tortoise was studied by gamma- and pulse-radiolysis. In the first step, one tryptophan side chain is oxidized to indolyl free radical, which is produced quantitatively. As shown already, the indolyl radical subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction. However this reaction is not total and its stoichiometry depends on the protein. Rate constants also vary between proteins, from 120 x s(-1) to 1000 x s(-1) at pH 7.0 and room temperature [extremes are hen and turkey egg white (120 x s(-1)) and human milk (1000 x s(-1))]. In hen and turkey egg white lysozymes we show that another reactive site is the Asn103-Gly104 peptidic bond, which gets broken radiolytically. Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme. Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine. Tyr23 appears not to be involved in the process. Thus new features of long-range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis.     

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.     

Differential Patterns of Bacteriolytic Activities in Potato in Comparison to Bacteriophage T4 and Hen Egg White Lysozymes

A comparison of the enzymatic activities of endogenous potato bacteriolytic enzymes with bacteriophage T4 and hen egg white lysozyme has been performed. Using Erwinia carotovora atroseptica and Pseudomonas solanacearum as substrates in, comparison to Micrococcus lysodeikticus a differential pattern of bacteriolytic activities could be detected. The expression pattern of endogenous potato lysozymes suggests that their functional activity against phytopathogenic bacteria in planta is unlikely. Antibacterial activities in transformed, T4 lysozyme expressing and non–transformed potato plants are evaluated.     

The amino acid sequence of equine milk lysozyme

The amino acid sequence of equine milk lysozyme has been elucidated. The study involves the determination of the sequence of the N-terminal region of the whole protein, cyanogen bromide fragments, tryptic and chymotryptic peptides and fragments produced by chemical cleavage after tryptophan residues. The protein consists of a single chain of 129 amino acid residues and has a Mr of 14647. While equine milk lysozyme has the essential features of a c(chick)-type lysozyme, there is only 51% sequence homology with human milk lysozyme and 50% with domestic hen egg white lysozyme. Some of the implications of the large number of differences are discussed.     

X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.     

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture,model of the Escherichia coli cytoplasmic membrane

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.     

A simple technique for the immobilization of lysozyme by cross-linking of hen egg white foam

A novel technique has been described for the immobilization of lysozyme, naturally present in hen egg white by cross-linking the egg white foam with glutaraldehyde. This technique results in a mechanically stable and porous matrix exhibiting about 6-times the lytic activity against Micrococcus lysodeikticus cells, as compared to the unfoamed matrix. Foamed egg white matrix can be used for the continuous lysis of bacterial cells     

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.     

Structural analysis of an insect lysozyme exhibiting catalytic efficiency at low temperatures

Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.     

Protein-protein recognition and interaction hot spots in an antigen-antibody complex: free energy decomposition identifies "efficient amino acids"

The molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method was applied to the study of the protein-protein complex between a camelid single chain variable domain (cAb-Lys3) and hen egg white lysozyme (HEL), and between cAb-Lys3 and turkey egg white lysozyme (TEL). The electrostatic energy was estimated by solving the linear Poisson-Boltzmann equation. A free energy decomposition scheme was developed to determine binding energy hot spots of each complex. The calculations identified amino acids of the antibody that make important contributions to the interaction with lysozyme. They further showed the influence of small structural variations on the energetics of binding and they showed that the antibody amino acids that make up the hot spots are organized in such a way as to mimic the lysozyme substrate. Through further analysis of the results, we define the concept of "efficient amino acids," which can provide an assessment of the binding potential of a particular hot spot interaction. This information, in turn, can be useful in the rational design of small molecules that mimic the antibody. The implications of using free energy decomposition to identify regions of a protein-protein complex that could be targeted by small molecules inhibitors are discussed.     

Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose

The three-dimensional structure of the lytic transglycosylase from bacteriophage lambda, also known as bacteriophage lambda lysozyme, complexed to the hexasaccharide inhibitor, hexa-N-acetylchitohexaose, has been determined by X-ray crystallography at 2.6 A resolution. The unit cell contains two molecules of the lytic transglycosylase with two hexasaccharides bound. Each enzyme molecule is found to interact with four N-acetylglucosamine units from one hexasaccharide (subsites A-D) and two N-acetylglucosamine units from the second hexasaccharide (subsites E and F), resulting in all six subsites of the active site of this enzyme being filled. This crystallographic structure, therefore, represents the first example of a lysozyme in which all subsites are occupied, and detailed protein-oligosaccharide interactions are now available for this bacteriophage lytic transglycosylase. Examination of the active site furthermore reveals that of the two residues that have been implicated in the reaction mechanism of most other c-type lysozymes (Glu35 and Asp52 in hen egg white lysozyme), only a homologous Glu residue is present. The lambda lytic transglycosylase is therefore functionally closely related to the Escherichia coli Slt70 and Slt35 lytic transglycosylases and goose egg white lysozyme which also lack the catalytic aspartic acid.     

An NMR and MD study of complexes of bacteriophage lambda lysozyme with tetra- and hexa-N-acetylchitohexaose

The X-ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa-N-acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E′F′ sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide. ¹H^N and ¹⁵N chemical shifts of λ lysozyme resonances were followed during NAG4/NAG6 titrations. The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme-inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme-peptidoglycan complex.     

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.     

Isolation of lysozyme on chitosan.

Lysozyme has been immobilized on chitosan, a polyaminosaccharide, without using any intermediate reagent. The best pH conditions for operating the chitosan columns have been determined and the best eluting agent was found to be a 2% solution of propylamine. The lysozyme activity was determined after reacting lysozyme with the product of glycolchitin and Remazol Brilliant Blue R. The recovery of lysozyme from chicken egg white yields lysozyme with 55% activity.