Structure Of Hen Egg White Lysozyme Research Articles

A new dimension in structural biology: fully fledged high-pressure macromolecular crystallography

The amount of accurate crystallographic data currently available on macromolecular structures at high pressure is extremely limited, mainly due to the lack of appropriate instrumentation and X-ray sources. A technical breakthrough has been achieved with a set-up at the ESRF ID30 beamline equipped with a diamond anvil cell and a large imaging plate, and taking advantage from undulators providing a quasi-plane wave of ultrashort wavelength X-rays. The accessible pressure range is increased by nearly one order of magnitude with respect to beryllium cells. A nearly perfect long-range order is preserved in protein and virus crystals compressed below the denaturation pressure. The quality of diffraction data collected at high pressure can meet usual standards, especially in the case of high symmetry space groups. An overview of main results and ongoing studies is given, including the 7 kbar structure of hen egg-white lysozyme refined at 1.6 Å resolution and structural investigations of cowpea mosaic virus (CPMV), the first macromolecular assembly investigated at high pressure by single crystal X-ray diffraction. The ordering effect of high pressure on the molecular packing of disordered P23 crystals was highlighted. The 2.8 Å resolution structure of CPMV at 3.3 kbar was fully refined using high completeness data collected on pressure-ordered I23 crystals.

Pressure-dependent Changes in the Solution Structure of Hen Egg-white Lysozyme

The “rules” governing protein structure and stability are still poorly understood. Important clues have come from proteins that operate under extreme conditions, because these clarify the physical constraints on proteins. One obvious extreme is pressure, but so far little is known of the behavior of proteins under pressure, largely for technical reasons. We have therefore developed new methodology for calculating structure change in solution with pressure, using NMR chemical shift changes, and we report the change in structure of lysozyme on going from 30 bar to 2000 bar, this being the first solution structure of a globular protein under pressure. The α-helical domain is compressed by approximately 1%, due to tighter packing between helices. The interdomain region is also compressed. By contrast, the β-sheet domain displays very little overall compression, but undergoes more structural distortion than the α-domain. The largest volume changes tend to occur close to hydrated cavities. Because isothermal compressibility is related to volume fluctuation, this suggests that buried water molecules play an important role in conformational fluctuation at normal pressures, and are implicated as the nucleation sites for structural changes leading to pressure denaturation or channel opening.

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study.

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.

Stokes-flow computation of the diffusion coefficient and rotational diffusion tensor of lysozyme, a globular protein

Based on a closed surface of triangles fitted to atomic coordinates determined crystallographically, Brune and Kim [Proc. Natl. Acad. Sci. USA 90, 3835–3839 (1993)] proposed a boundary-element Stokes-flow technique for ab initio computation of a translational diffusion coefficient and the rotational diffusion tensor Dr of globular proteins. They applied their approach to atomic coordinates for a tetragonal structure of hen egg-white lysozyme, and reported that computed values of a translational diffusion coefficient and Dr=tr(Dr)/3 agreed well with experiment. After establishing the identity between the infinite-dilution tracer diffusion coefficient of the protein macroion (D+ for lysozyme cation) and the “translational diffusion coefficient” computed by Brune and Kim, we adopt a somewhat different computational approach and show how convergence of D+ and Dr for tetragonal lysozyme depends on two computational parameters characterizing the fidelity of the geometric approximation to the protein surface and two others characterizing the accuracy of the Stokes-flow computations. We then compute D+ and Dr for lysozyme using atomic coordinates for the triclinic crystal structure, three structures determined by nuclear magnetic resonance spectroscopy in the liquid phase (presumably corresponding more closely to in vivo structures), the solvated tetragonal structure (with 108 water molecules) considered by Brune and Kim, and a “dry” version of the same structure. These computations show that D+ and Dr computed for all of the dry crystal structures are in excellent agreement with those for the liquid-phase conformations. Values of D+ and Dr computed for the solvated structure are lower, consistent with the larger volume and area of the corresponding polyhedral surface. We also show that several choices of the origin of the force system [discussed by Brenner, J. Colloid Interface Sci. 23, 407–436 (1967)] give rise to nearly identical translational diffusion coefficients. Finally, we show how to estimate the thickness of the “solvation shell” contributing to the hydrodynamic resistance of the protein cation, and use the binary Nernst–Hartley equation to then estimate the effective cation charge at the two pH values at which the binary diffusion coefficient has been accurately measured in recent interferometric experiments.

Structural effects of monovalent anions on polymorphic lysozyme crystals.

Understanding direct salt effects on protein crystal polymorphism is addressed by comparing different crystal forms (triclinic, monoclinic, tetragonal and orthorhombic) for hen, turkey, bob white quail and human lysozymes. Four new structures of hen egg-white lysozyme are reported: crystals grown in the presence of NapTS diffracted to 1.85 A, of NaI to 1.6 A, of NaNO(3) to 1.45 A and of KSCN to 1.63 A. These new structures are compared with previously published structures in order to draw a mapping of the surface of different lysozymes interacting with monovalent anions, such as nitrate, chloride, iodide, bromide and thiocyanate. An analysis of the structural sites of these anions in the various lysozyme structures is presented. This study shows common anion sites whatever the crystal form and the chemical nature of anions, while others seem specific to a given geometry and a particular charge environment induced by the crystal packing.

Molecular dynamics simulation of hen egg white lysozyme: A test of the GROMOS96 force field against nuclear magnetic resonance data

Biomolecular force fields for use in molecular dynamics (MD) simulations of proteins, DNA, or membranes are generally parametrized against ab initio quantum-chemical and experimental data for small molecules. The application of a force field in a simulation of a biomolecular system, such as a protein in solution, may then serve as a test of the quality and transferability of the force field. Here, we compare various properties obtained from two MD simulations of the protein hen egg white lysozyme (HEWL) in aqueous solution using the latest version, GROMOS96, of the GROMOS force field and an earlier version, GROMOS87+, with data derived from nuclear magnetic resonance (NMR) experiments: NOE atom–atom distance bounds, 3JHNα-coupling constants, and backbone and side-chain order parameters. The convergence of these quantities over a 2-ns period is considered, and converged values are compared to experimental ones. The GROMOS96 simulation shows better agreement with the NMR data and also with the X-ray crystal structure of HEWL than the GROMOS87+ simulation, which was based on an earlier version of the GROMOS force field. Proteins 2000;40:145–153. © 2000 Wiley-Liss, Inc.

Molecular dynamics simulation of hen egg white lysozyme: A test of the GROMOS96 force field against nuclear magnetic resonance data

Biomolecular force fields for use in molecular dynamics (MD) simulations of proteins, DNA, or membranes are generally parametrized against ab initio quantum-chemical and experimental data for small molecules. The application of a force field in a simulation of a biomolecular system, such as a protein in solution, may then serve as a test of the quality and transferability of the force field. Here, we compare various properties obtained from two MD simulations of the protein hen egg white lysozyme (HEWL) in aqueous solution using the latest version, GROMOS96, of the GROMOS force field and an earlier version, GROMOS87+, with data derived from nuclear magnetic resonance (NMR) experiments: NOE atom-atom distance bounds, (3)J(HNalpha)-coupling constants, and backbone and side-chain order parameters. The convergence of these quantities over a 2-ns period is considered, and converged values are compared to experimental ones. The GROMOS96 simulation shows better agreement with the NMR data and also with the X-ray crystal structure of HEWL than the GROMOS87+ simulation, which was based on an earlier version of the GROMOS force field.

23aC3 Crystal structure of hen egg-white lysozyme within high magnetic-field environment

23aC3 Crystal structure of hen egg-white lysozyme within high magnetic-field environment

Succinimide and isoaspartate residues in the crystal structures of hen egg-white lysozyme complexed with tri- N-acetylchitotriose

The isomerization of Asp101 to isoaspartate autocatalytically proceeds via a succinimide intermediate in hen egg-white lysozyme at a mildly acidic condition. The crystal structures of succinimide and isoaspartate forms of the lysozyme proteins, each complexed with a tri- N -acetylchitotriose ligand, have been determined at 1.8 Å resolution, and distinctively elucidate coplanar cyclic aminosuccinyl and β-linked isoaspartyl residues. Compared with the liganded native protein with normal Asp101, succinimide 101 protrudes toward the ligand, and isoaspartate 101 extends away from the ligand. The formations of these residues caused the loss of three hydrogen-bonds between the ligand and the side-chains of Asp101 and Asn103 along with 0.5 Å displacement of the ligand location.

Distribution analysis of the variation of B-factors of X-ray crystal structures; temperature and structural variations in lysozyme.

The B-factor (isotropic temperature factor) data for X-ray structures of hen egg-white lysozyme from the study of Young et al. (Young, Dewan, Nave, and Tilton J. Appl. Cryst. 1993, 26, 309-319) potentially contain information about the relative contributions of static and dynamic variation to these factors. The six structures of the protein were obtained at two widely different temperatures (100 and 298 K), with two crystal forms (monoclinic and tetragonal) and other experimental differences. In addition, the monoclinic lysozyme crystals with two molecules per asymmetric unit allow direct examination of variation between structures determined under identical conditions at both temperatures. The B-factors from these structures all have complex distribution functions as might be expected considering all of the influences that these values must reflect. The empirical cumulative distribution functions (eCDF's) of these data show that they are representative of complex, multicomponent distributions. Distribution analysis using the DANFIP procedure (Wampler, Anal. Biochemistry 1990, 186, 209-218) of the data sets reveals that they can be modeled as four to six Gaussian subpopulations, that these subpopulations do not correlate with specific atom types, specific amino acid residues or fixed locations in the structure. While they do seem to correlate with localized groupings of atoms, these grouping vary from structure to structure even within the same crystal under the same conditions. Temperature seems to have a global effect in this case, but it is clear that other factors including experimental error influence the distribution of B-factors within a given structure. This analysis also helps explain the oft observed lack of atomic level correlation between experimental B-factors and calculated mean square displacements from molecular dynamics simulations.

Characterization of Conformational Preferences in a Partly Folded Protein by Heteronuclear NMR Spectroscopy: Assignment and Secondary Structure Analysis of Hen Egg-White Lysozyme in Trifluoroethanol

2D and 3D heteronuclear NMR methods have been used to characterize the structure of hen egg-white lysozyme in a partially folded state using uniformly 15N-labeled protein. This state is formed by the denaturation of the protein in 70% trifluoroethanol (TFE)/30% water (v/v) and is characterized by substantial helical secondary structure in the absence of extensive tertiary interactions. 15N-filtered 3D NOESY and TOCSY experiments have allowed the sequential assignment of resonances for all but 2 of the 126 main chain amide nitrogen atoms and of the majority of main and side chain proton resonances. The conformation of the polypeptide chain was characterized by analysis of the pattern of NOEs, H alpha chemical shift perturbations, 3J(HN, H alpha)-coupling constants, and hydrogen exchange protection. These NMR parameters are highly complementary and are consistent with a model for the TFE state in which six regions of the polypeptide chain are substantially ordered in helical conformations. The structure in different regions however, shows different levels of persistency. Five of the helices exhibit significant protection of amide hydrogens against exchange with solvent and are located in regions of the polypeptide which are helical in the native state. By contrast, helical structures of greater flexibility are observed both as extensions to the native-like helices and as a nonnative structure in the region of the molecule which forms the C-terminal part of the beta-sheet in the native state. No specific structural preferences are detected in regions corresponding to the long loop and to the N-terminal part of the beta-sheet of native lysozyme. A combination of local features of the polypeptide chain, including the predicted propensities of residues for helix formation and for their participation in N- and C-terminal helix capping interactions, allows the conformational behavior of the polypeptide chain of hen lysozyme to be rationalized for this partially folded state. The analysis implies that the nonnative structures are a result of interactions which are local to the polypeptide chain. These, and the highly persistent native-like structures, give insight into species which form early during folding.

A structural basis for the interaction of urea with lysozyme.

The effect of urea on the crystal structure of hen egg-white lysozyme has been investigated using X-ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type II crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 A resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 A. Conformational changes are limited to flexible surface loops (Thr 69-Asn 74, Ser 100-Asn 103), the active site loop (Asn 59-Cys 80), and the C-terminus (Cys 127-Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N-acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.

Characteristics of the epitope of protein-reactive anti-peptide antibodies.

The specificity of hen egg-white lysozyme (HEL)-reactive rabbit antibodies induced by the peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) of HEL was clarified by analyzing their cross-reactions with various avian lysozymes and their reaction with synthetic peptides (sequences 59-82) in which alanine was substituted for the amino acid at certain positions. The Arg-68 residue of HEL plays a dominant role in the binding, while Gly-71, Ser-72, Arg-73, and Pro-79 also contribute to the binding of two anti-Ploop I.II antibodies (rabbit number 125 and 126). These residues, although remote in sequence, are grouped together in the crystal structure of HEL and may form an area of contact with the antibody. Contributions by Trp-63, Ile-78, and Asn-77 to the binding of the two antibodies to HEL were excluded. These results support the idea that the anti-Ploop I.II antibodies recognize a conformational type of epitope which is similar to that of native HEL. The immunogenicity of the reduced and alkylated form of Ploop I.II was also tested, but it failed to induce an HEL-reactive antibody.

Comparison of structures of dry and wet hen egg-white lysozyme molecule at 1.8 Å resolution

A high resolution structure of hen egg-white lysozyme containing 36 ± I mol H 2O per mol of protein has been obtained using triclinic (P 1) crystals cross-linked with glutaraldehyde. Analysis of dehydration-induced structural changes has revealed displacement in relative position of domains and numerous small displacements in positions of individual atoms with r.m.s. deviation of main atoms 0.60 Å, and that of all atoms 0.97 Å. An increase in the average packing density of atoms in dry lysozyme by 4–6% seems to be the most probable reason for the loss of its activity and mobility.

Crystal structure of hen egg-white lysozyme at a hydrostatic pressure of 1000 atmospheres

The crystal structure of tetragonal hen egg-white lysozyme at a hydrostatic pressure of 1000 atmospheres has been determined by X-ray diffraction to a nominal resolution of 2 Å. The crystals, originally grown in 0.83 m-NaCl, had to be transferred to 1.4 m-NaCl to prevent crystal cracking at 300 to 400 atm. The a and b axes of the unit cell contracted by 0.6%, whilst the c axis increased by 0.1%. The unit cell volume contracted by 1.1%. Both the 1 atm and the 1000 atm structures were refined by restrained least-squares to yield final R factors of 14.9% in each case. Since the data were collected by an accurate difference protocol, the change in structure is considered to be more accurate than the absolute structure. The probable accuracy of the atomic shifts is shown to be ±0.06 Å. The estimated volume decrease of the whole molecule corresponded to an isothermal compressibility of 4.7 × 10 −3 kbar −1. The contraction was non-uniformly distributed. Domain 2 (residues 40 to 88) was essentially incompressible, whilst domain 1 (residues 1 to 39, 89 to 129) had a compressibility of 5.7 × 10 −3 kbar −1. The interdomain region was also compressible. The average B factor decreased about 1 Å 2 at 1000 atm, but there was a wide range of decreases and increases in individual values. The pressure-induced deformation was analyzed with difference distance matrices. The beta-sheet (residues 42 to 60) and helix 2 (residues 24 to 36) were deformed the least under pressure. The other helices were more deformed and one loop region (residues 61 to 87) actually appeared to expand. The main-chain atoms of the beta-sheet and helix 2 were used to perform a least-squares superposition of the 1 atm and 1000 atm models. The root-mean-square pressure-induced shift for all atoms was 0.2 Å, with a few atoms moving more than 1 Å. There was no evidence for co-ordinated movement about the hinge axis defined by alpha carbon atoms 38 and 97. The 1 atm and 1000 atm refined structures included 151 and 163 ordered water molecules, respectively. The changes in these ordered water molecules and the mean compressibility of all of the solvent in the crystal will be described elsewhere.

Amino acid substitutions far from the active site of bacteriophage T4 lysozyme reduce catalytic activity and suggest that the C-terminal Lobe of the enzyme participates in substrate binding

As part of a systematic study of the effect of single amino acid substitutions on the structure, function and stability of the lysozyme from bacteriophage T4, we have characterized a mutant lysozyme for which the catalytic activity is reduced to 4% that of the wild-type enzyme, yet the location of the amino acid substitution is on the far side of the molecule, 25 A from the active site. Genetic and chemical analysis show that the mutant lysozyme differs from wild-type only in the replacement of Glu128 by a lysine residue. Crystallographic analysis confirms the location of the amino acid substitution, and also shows that the replacement of Glu128, which is on the surface of the protein, causes very little change in the three-dimensional structure of the lysozyme molecule. Thermodynamic analysis shows that the stability of the mutant lysozyme is comparable with that of native lysozyme. These results suggest that Glu128 participates directly in substrate binding and/or catalysis, and argue against the possibility that the low activity of the mutant enzyme is due to an indirect effect such as destabilization of the lysozyme molecule, or modification of its three-dimensional structure. Glu128 is located in the part of the carboxy-terminal domain of phage lysozyme that has no counterpart in the structure of hen egg-white lysozyme. We suggest that the role of this C-terminal domain is to bind the peptide cross-link that connects neighboring saccharide strands within the cell walls of Escherichia coli . This postulate is consistent with the known differences in specificity between phage lysozyme and hen egg-white lysozyme, and is also compatible with the activity of native and mutant phage lysozymes toward different bacterial cell walls.

Dynamic information from protein crystallography: An analysis of temperature factors from refinement of the hen egg-white lysozyme structure

A preliminary analysis is presented of whether the isotropic temperature factors derived from refinement of the tetragonal crystal structure of hen egg-white lysozyme at 2 Å resolution can be interpreted in terms of molecular motion. If the contributions to the temperature factors from experimental errors, crystal disorder and imperfections of the molecular model are neglected, the apparent thermal motion is found to be compatible with the pair of molecules that have the strongest interactions in the crystal either moving as a rigid-body with libration about a common axis, or vibrating in an intramolecular mode with the principal amplitudes radial to this axis. There is only weak evidence for hingebending, i.e. that the two lobes of lysozyme vibrate so that their separation varies. Side-chains that are exposed to the solvent and some segments of the main polypeptide chain (including residues 70–73, 82–84, 101–103) have greater apparent thermal motion than the remainder of the molecule. Although this analysis at a single temperature cannot establish whether thermal motion or static disorder (including conformational variability) underlies the observed effects, it suggests that the accurate determination of temperature factors will be useful in detailed studies of the dynamic properties of macromolecules.

The structure of phage T4 lysozyme in solution noticeably differs from its crystalline structure

In the preceding paper [l] it was shown by the method of large-angle diffuse X-ray scattering [2,3] that the structure of bovine pancreatic ribonuclease and that of its complex with an inhibitor in solution coincide with their crystalline structures(on a distance scale 2 10 A). Also it was shown [2] that hen egg white lysozyme structure in solution differs from its crystalline structure by a small opening of the substrate-binding cleft, the binding of the competitive inhibitor stabilizing the closed state of the cleft observed in the crystalline structure. Sperm whale myoglobin structure in solution also differs from its crystalline structure, the differences being interpreted as changes of the GH hairpin position relative to the remaining part of the molecule [4] . In all probability in both cases we are dealing with fluctuations in relative positions of large masses of a protein molecule which are frozen in the crystalline structure but which manifest themselves in solution. The possibility of such fluctuations was demonstrated by direct model calculations for hen egg-white lysozyme [2]. Here the above-mentioned method has been used to study phage T4 lysozyme. The crystalline structure of this protein is characterized by the closed substratebinding cleft, therefore ‘in order to allow the substrate to enter, the enzyme would have to undergo a fairly substantial conformational change’ [6] . Our results provide evidence of a noticeable difference between

Facile assignment of disulfide bonds in proteins through detection of cystine-containing peptides in peptide maps

A new procedure for the facile assignment of the positions of disulfide bonds in proteins of known amino acid sequence is described. Hen egg-white lysozyme was subjected to proteolytic digestion by pepsin and chymotrypsin. A peptide map of the enzymatic hydrolysate was prepared. Seven disulfide-containing peptides were detected after reduction with sodium borohydride and treatment with 5,5′-dithiobis(2-nitrobenzoic acid) [DTNB]. The cystine-containing peptides were eluted, hydrolysed, and subjected to amino acid analysis. By comparison of their amino acid compositions with the known sequence of lysozyme, the positions of all four disulfide bonds could be assigned. The established covalent structure of hen egg-white lysozyme was readily confirmed.

Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution.

Structure of Hen Egg-White Lysozyme: A Three-dimensional Fourier Synthesis at 2 A Resolution