Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol

Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied by using CD and amide proton exchange monitored by two-dimensional 1H NMR. The far- and near-UV CD spectra of the protein showed that the secondary and tertiary structures of lysozyme in glycerol were similar to those in water. Thermal melting of lysozyme in glycerol followed by CD spectral changes indicated unfolding of the tertiary structure with a Tm of 76.0 +/- 0.2 degreesC and no appreciable loss of the secondary structure up to 85 degreesC. This is in contrast to the coincident denaturation of both tertiary and secondary structures with Tm values of 74.8 +/- 0.4 degreesC and 74.3 +/- 0.7 degreesC, respectively, under analogous conditions in water. Quenched amide proton exchange experiments revealed a greater structural protection of amide protons in glycerol than in water for a majority of the slowly exchanging protons. The results point to a highly ordered, native-like structure of lysozyme in glycerol, with the stability exceeding that in water.     

The origin of the alpha-domain intermediate in the folding of hen lysozyme

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with quenched-flow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to compare the refolding kinetics of hen egg-white lysozyme at 20 degrees C and 50 degrees C. At 50 degrees C there is clear evidence for distinct fast and slow refolding populations, as observed at 20 degrees C, although folding occurs significantly more rapidly. The folding process is, however, substantially more cooperative at the higher temperature. In particular, the transient intermediate on the major refolding pathway at 20 degrees C, having persistent native-like structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labelling at 50 degrees C. In addition, the characteristic maximum in negative ellipticity and the minimum in fluorescence intensity observed in far UV CD and intrinsic fluorescence experiments at 20 degrees C, respectively, are not seen at 50 degrees C. Addition of 2 M NaCl to the refolding buffer at 50 degrees C, however, regenerates both the hydrogen exchange and optical properties associated with the alpha-domain intermediate but has no significant effect on the overall refolding kinetics. Together with previous findings, these results indicate that non-native interactions within the alpha-domain intermediate are directly responsible for the unusual optical properties observed during refolding, and that this intermediate accumulates as a consequence of its intrinsic stability in a folding process where the formation of stable structure in the beta-domain constitutes the rate-limiting step for the majority of molecules.     

Identification of tryptophan 62 as an ozonization-sensitive residue in hen egg-white lysozyme

The position of a single N'-formylkynurenine residue in ozone-inactivated hen egg-white lysozyme [EC 3.2.1.17] was determined by two methods. One involved identification of an amino-terminus of the C-peptide obtained by selective cleavage with hydrazine of the kynurenyl peptide linkage in oxidized lysozyme. The other was to analyze a tryptic peptide containing kynurenine in the modified enzyme. Both methods showed that tryptophan 62 in lysozyme was so sensitive to ozone as to be selectively oxidized to N'-formylkynurenine with concomitant loss of the lytic activity.     

Antibody response of murine B1 cells to hen eggwhite lysozyme

Cell transfer studies were performed to investigate the ability of murine peritoneal B1 cells to produce specific IgG antibody against the T-dependent protein antigen, hen eggwhite lysozyme (HEL). Peritoneal cells (PeC) from normal BALB/c mice were transferred into newborn, allotype-congenic, C.B-17 severe combined immunodeficient (SCID) mice alone or together with splenic T cells from HEL-primed C.B-17 mice. After immunization with HEL, only those mice that received both PeC and primed T cells produced HEL-specific IgG of the PeC donor allotype. To identify the B cell subset responsible for antibody production, PeC were sorted before transfer into B1 and conventional B (B2) cell populations. It was found that transfer of purified B1 cells plus primed T cells resulted in high levels of IgG1 anti-HEL in approximately half of the SCID recipients, while mice receiving B2 cells produced little detectable antibody. The responses consisted primarily of IgG1 kappa anti-HEL, with no significant IgM or lambda-bearing components. Seventeen HEL-specific hybridomas of BALB/c origin, i.e., derived from the B1 cell donor, were obtained from reconstituted SCID mice after various periods of immunization and analyzed for fine specificity using a panel of avian lysozymes. All but one of the B1 cell-derived mAbs recognized an HEL epitope not present on the closely related bobwhite quail lysozyme (differing from HEL at only 4 of 129 amino acid positions). While IEF analyses demonstrated the presence of extensive clonotypic diversity, the epitope specificity pattern, which is rare among B2-cell-derived antibodies, suggests that the B1 cell recognition repertoire for HEL is severely limited.     

Non-native interactions in protein folding intermediates: molecular dynamics simulations of hen lysozyme

Molecular dynamics simulations of protein denaturation can complement and extend experimental studies of protein folding by providing atomic-level structural information about conformational transitions and any conformational states along the unfolding pathway. Previous unfolding simulations of hen egg-white lysozyme have resulted in intermediate structures with an unfolded alpha-domain and a structured beta-domain, which is inconsistent with experiment. In contrast, the beta-domain unfolded first in the two simulations presented here leaving a structured alpha-domain. Following this, intermediate states were identified that differ with respect to the packing of the helices and the elements of non-native structure adopted. The non-native structure is critical for explaining many of the experimental observations. Overall, the pooled ensemble of these intermediates is in agreement with the experimental data for the major kinetic intermediate, suggesting that the kinetic intermediate may be made up of distinct, but rapidly interconverting, partially folded conformations distinguished primarily by differences in helix packing.     

Studies on delayed-type hypersensitivity to hen egg-white lysozyme. I. Peptide fragments of lysozyme inducing delayed-type hypersensitivity

Eleven peaks were separated by Carboxymethyl-cellulose column chromatography of peptic digest of lysozyme. Being stronger in antigenic activity two peaks of them, P-3 and P-9, were selected and purified further respectively by Amberlite IRC-50 and Sephadex G-50 column chromatography. As the results, PP-3 and PP-9 were obtained each as a single peak. For estimation of their capacities to induce delayed-type hypersensitivity, the antigen-induced 3H-thymidine incorporation, the migration inhibition of peritoneal cells and the delayed-type skin reaction were tested in guinea pigs immunized with native lysozyme or any of its fractions. PP-9 was almost as active as intact lysozyme in these capacities. On the other hand, PP-3 showed a slight inhibition of migration of peritoneal cells and no stimulation of 3H-thymidine incorporation into the lymph node cells. Moreover, the delayed-type skin reaction elicited by PP-3 was always weaker than that elicited by PP-9. Guinea pigs immunized with either PP-3 or PP-9 were also tested for these reactions. PP-9 and native lysozyme elicited these reactions in guinea pigs immunized with PP-9, but PP-3 did not. On the other hand, PP-3 and lysozyme elicited these reactions in those immunized with PP-3, but PP-9 did not. The possibility of recognition of two functionally different areas, one for production of the circulating antibody and the other for induction of delayed-type hypersensitivity, on the lysozyme molecule was discussed.     

Characterization of collapsed states in the early stages of the refolding of hen lysozyme

Early conformational states in the refolding of hen lysozyme from guanidine hydrochloride have been characterized by measuring both the fluorescence and the solvent exchange properties of tryptophan side chains. The indole proton occupancies indicate that at pH 5.5, 25 degrees C, half the protection against pulse labeling occurs in the dead time (4 ms) of the experiment, with the remaining protection developing with a time constant of 55 ms. Comparison of these data with the protection kinetics of backbone amides and with the fluorescence data provides evidence for hydrophobic collapse involving incorporation of tryptophan residues in a solvent-excluded state in advance of stable secondary structure formation. Analysis of the pH dependence of the indole hydrogen exchange protection is consistent with two or more structurally distinct collapsed states, and indicates that the generation of a correctly folded compact hydrophobic core is a key precursor to the formation of persistent native-like structure during refolding.     

The folding process of hen lysozyme: a perspective from the ''new view''

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer.     

Effects of subsite alterations on substrate-binding mode in the active site of hen egg-white lysozyme

The primary sequence of the low-molecular-mass cadmium-binding protein metalloprotein II of Nereis diversicolor (Hediste diversicolor, recent denomination) has been determined. This protein is composed of 119 amino acids and has 80.8% identity with the N. diversicolor myohemerythrin [Takagi, T. & Cox, J. A. (1991) FEBS Lett. 285, 25-27]. The fact that iron, which normally binds to myohemerythrin, is not found to be associated with the cadmium-binding protein metalloprotein II in cadmium-exposed animals could be the result of the complete abolition of the iron-binding capacity of the protein due to the binding of cadmium.     

Kinetics of secondary structure recovery during the refolding of reduced hen egg white lysozyme

We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 M guanidinium chloride, 60 microM oxidized glutathione, and 20 microM reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi- or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.     

Analysis of the stabilization of hen lysozyme by helix macrodipole and charged side chain interaction

In the N-terminal region of the alpha-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Asp18/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asn18/Asn27 (18N/27N), Asn18/Asp27 (18N/27D), and Asp18/Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Asp18 and the amide group at the N1 position in the alpha-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Asp18 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the protein.     

Molecular dynamics simulations of peptide fragments from hen lysozyme: insight into non-native protein conformations

Molecular dynamics simulations of four peptides taken from the hen lysozyme sequence have been used to generate models for non-native protein conformations. Comparisons between the different peptides and with experimental data for denatured lysozyme and peptide fragments provides insight into the characteristics of the conformational ensembles populated in these non-native states and the dependence of their structural features on the amino acid sequence. For the denatured conformers populated local contacts dominate in determining the properties observed in the trajectories, all four peptides showing similar characteristics. These include a significant increase in the number of main-chain O(i)-NH(i+2) hydrogen bonds and hydrogen bonds involving side-chain groups, this increase compensating to a large extent for the loss of hydrogen bonds involved in helical or beta-sheet secondary structure in the native fold, and the generation of a population of collapsed states with local clusterings of hydrophobic groups. The hydrophobic clusters enable at least partial burial of many side-chains exposed by the loss of tertiary contacts on denaturation and provide models that may explain the experimentally observed protection of amides from hydrogen exchange and the existence of residual secondary structure in non-native species of lysozyme. The results suggest that this approach has an important role to play in aiding the interpretation of experimental data for conformationally disordered non-native states of proteins.     

State of imidazole side chain of hen lysozyme modified with histamine and Japanese quail lysozyme. A study by immobilized metal ion affinity chromatography

Hen lysozyme modified with histamine (HML) and Japanese quail lysozyme (JQL) were treated with immobilized metal ion affinity chromatography to analyze the states of their imidazole groups. When Ni(II) was used as the metal ion immobilized, JQL was strongly retained in a Ni(II)-chelating Sepharose column, while hen lysozyme and HML were hardly retained in the same column. All of these lysozymes have a histidine imidazole group at the 15th position, while JQL has an additional histidine imidazole group at the 103rd position and HML has an additional imidazole group covalently attached to Asp101. Thus, I concluded that the imidazole group at the 103rd position of JQL is exposed to the solvent and recognized by the metal ion, but that the imidazole group attached to Asp101 in HML is localized to a hydrophobic region and not recognized by the metal ion.     

Structure of warfarin in solution

Warfarin in solution is shown to consist of three interconverting tautomeric structures, two of which are cyclic diastereomeric hemiketals, while the third and minor component is the open-chain intermediate form. The configurations of all the tautomers as well as the major conformations of the cyclic tautomers are assigned. The assignments are supported by comparison with the chemical shift and coupling constant parameters of structurally fixed model compounds.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

Formation of delta1-acetoxytryptophan-62 in the oxidation of tryptophan-62 of hen egg-white lysozyme by N-bromosuccinimide in acetate buffer

The reaction of equimolar amounts of N-bromosuccinimide and hen egg-white lysozyme in acetate buffer, under the conditions of Hayashi et al. (Hayashi, K., Imoto, T., Funatsu, G., and Funatsu, M. (1965), J. Biochem. (Tokyo) 58, 227), yields a protein mixture that has a time-dependent 13C-NMR spectrum. The initial natural-abundance 13C-NMR spectrum indicates the presence of about equal amounts of [oxindolealanine-62]lysozyme and [delta1-acetoxytryptophan-62]lysozyme. The latter converts to [oxindolealanine-62]lysozyme with a half-life of about 2 days at 25 degrees C and pH 3.9. Two observations indicate that the source of the acetyl group of delta1-acetoxytryptophan-62 is the acetate buffer. First, the spectrum of a lysozyme sample treated with N-bromosuccinimide in the presence of [1-13C]acetate yields a very strong acetyl ester carbonyl resonance. The time dependence of the intensity of this resonance yields a half-life of 44 h for [delta1-acetoxytryptophan-62]lysozyme. Second, the initial natural-abundance 13C-NMR spectrum of a lysozyme sample treated with N-bromosuccinimide in the absence of acetate indicates essentially complete conversion of tryptophan-62 into oxindolealanine.     

Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: an X-ray analysis at 1.8-A resolution

Chemical modification of proteins has been and continues to be an important biochemical tool for the study of protein structure and function. One such type of approach has been the reductive methylation of lysine residues. In order to address the consequences of such methylation on the crystallization and structural properties of a protein, the three-dimensional structure of hen egg white lysozyme in which all lysine residues have been alkylated has been determined and refined to a nominal resolution of 1.8 A and a crystallographic R factor of 17.3%. Crystals used in the investigation were grown from 1.5-1.8 M MgSO4 and 50 mM Tris at pH 8.0 and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 30.6 A, b = 56.3 A, c = 73.2 A, and one molecule per asymmetric unit. It was not possible to grow crystals of the modified lysozyme under the conditions normally employed for the hen egg white protein. Overall, the three-dimensional structures of the native lysozyme and the modified protein are very similar with only two surface loops differing to any significant extent. Specifically, the positions of the alpha-carbons for these two forms of the protein, excluding the surface loops, superimpose with a root-mean-square value of 0.40 A. The magnitude of the structural changes observed between the modified an unmodified forms of lysozyme is similar to that seen when an identical protein structure is solved in two different crystalline lattices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Batch uptake of lysozyme: effect of solution viscosity and mass transfer on adsorption

In this study, solid-phase adsorption by macroporous and hyper-diffusive resins was investigated in a batch uptake adsorption system to quantify solid-phase diffusion rates as a function of bulk phase viscosity. The performance of chromatographic resins used for adsorption of proteins is dependent on several factors including solid and liquid-phase diffusivity, boundary layer mass transfer, and intraparticle mass transfer effects. Understanding these effects is critical to process development and optimization of both packed and fluidized bed adsorption systems. The macroporous resin used here was Streamline SP, and the hyper-diffusive resin was S-HyperD LS. Both have been frequently used in fluidized bed adsorption of proteins; however, factors that affect uptake rates of these media are not well quantified. Adsorption isotherms were well represented by an empirical fit of a Langmuir isotherm. Solid-phase diffusion coefficients obtained from simulations were in agreement with other models for macroporous and hyper-diffusive particles. S-HyperD LS in the buffer system had the highest uptake rate, but increased bulk phase viscosity decreased the rate by approximately 50%. Increases in bulk phase viscosity increased film mass transfer effects, and uptake was observed to be a strong function of the film mass transfer coefficient. Uptake by Streamline SP particles was slower than S-HyperD in buffer, due to a greater degree of intraparticle mass transfer resistance. The effect of increased film mass transfer resistance coupled with intraparticle mass transfer resistances at an increased bulk phase viscosity resulted in a decrease of 80% in the uptake rate by Streamline SP relative to S-HyperD.     

Protein interactions in solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen

The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH and change from positive to negative as the pH increases. All coefficients at high salt concentration are slightly negative and depend weakly on pH. For lysozyme, the coefficients always decrease with increasing electrolyte concentration. However, for chymotrypsinogen there is a cross-over point around pH 5.2, above which the virial coefficients decrease with increasing ionic strength, indicating the presence of attractive electrostatic interactions. The data are in agreement with Derjaguin-Landau-Verwey-Overbeek (DLVO)-type modeling, accounting for the repulsive and attractive electrostatic, van der Waals, and excluded volume interactions of equivalent colloid spheres. This model, however, is unable to resolve the complex short-ranged orientational interactions. The results of protein precipitation and crystallization experiments are in qualitative correlation with the patterns of the virial coefficients and demonstrate that interaction mapping could help outline new crystallization regions.