Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca(2+)-free form of the homologous alpha-lactalbumins forms an intermediate "molten globule" state. To understand this difference, we have produced a chimera of human lysozyme and bovine alpha-lactalbumin. In the synthetic gene of the former the sequence coding for amino acid residues 76-102 was replaced by that for bovine alpha-lactalbumin 72-97, which represents the Ca(2+)-binding loop and the central helix C. The chimeric protein, LYLA1, expressed in Saccharomyces cerevisiae was homogeneous on electrophoresis and mass spectrometry. Its Ca2+ binding constant was 2.50 (+/- 0.04) x 10(8) M-1, and its muramidase activity 10% of that of human lysozyme. One-dimensional NMR spectroscopy indicated the presence of a compact, well structured protein. From two-dimensional NMR spectra, main chain resonances for 118 of a total of 129 residues could be readily assigned. Nuclear Overhauser effect analysis and hydrogen-deuterium exchange measurements indicated the presence and persistence of all expected secondary structure elements. Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2+ form at 90 degrees C, whereas unfolding of the apo form occurred at 73 degrees C in the near-UV and 81 degrees C in the far-UV range. These observations illustrate that by transplanting the central part of bovine alpha-lactalbumin, we have introduced into human lysozyme two important properties of alpha-lactalbumins, i.e. stabilization through Ca2+ binding and molten globule behavior.
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PMID:A Ca(2+)-binding chimera of human lysozyme and bovine alpha-lactalbumin that can form a molten globule. 773 86

Four methods are compared to drive the unfolding of a protein: (1) high temperature (T-run), (2) high pressure (P-run), (3) by imposing a gradual increase in the mean radius of the protein using a penalty function added to the physical interaction function (F-run, radial force driven unfolding), and (4) by weak coupling of the difference between the temperature of the radially outward moving atoms and the radially inward moving atoms to an external temperature bath (K-run, kinetic energy driven unfolding). The characteristic features of the four unfolding pathways are analyzed in order to detect distortions due to the size or the type of the applied perturbation, as well as the features that are common to all of them. Hen egg white lysozyme is used as a test system. The simulations are analyzed and compared to experimental data like 1H-NMR amide proton exchange-folding competition, heat capacity, and compressibility measurements.
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PMID:Computational approaches to study protein unfolding: hen egg white lysozyme as a case study. 778 24

The bacteriophage lambda R gene has been isolated into an Escherichia coli expression system and the R gene product, a lysozyme, has been overexpressed and purified to homogeneity using an efficient purification procedure. A turbidimetric assay utilizing chloroform-treated E. coli cells has been optimized to assess the bacteriolytic activity of the purified enzyme. Using this assay, oligomers of beta (1 --> 4) N-acetyl-D-glucosamine at high concentrations were shown to inhibit lysozyme but were not cleaved by the enzyme. Differential scanning calorimetry revealed that the thermal denaturation of lysozyme was found to increase in the presence of (GlcNAc)3 and (GlcNAc)5. The lysozyme was also expressed in an E. coli strain auxotrophic for methionine, allowing for the incorporation of [methyl-13C]methionine into the enzyme. An alteration of the [1H-13C]HMQC NMR spectra of the labelled enzyme was observed in the presence of (GlcNAc)5. Commercially available nitrophenyl glycosides did not act as substrates for lambda lysozyme. The results indicate that lambda lysozyme has specific interactions with oligosaccharides of N-acetylglucosamine, but is incapable of hydrolyzing these sugars. The relevance of the structure of peptidoglycan to the activity of lambda lysozyme is discussed.
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PMID:Investigations of the interactions of saccharides with the lysozyme from bacteriophage lambda. 787 85

All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.
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PMID:Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR. 790 91

Mutations around His15 which lie far away from the active site, stimulated glycol chitin activity of lysozyme at physiological temperature. Del-Arg14His15 lysozyme, a mutant lysozyme whose Arg14 and His15 were deleted together, and has the highest activity among these mutant lysozymes, had a similar binding ability to a trimer of N-acetyl-glucosamine, a substrate analogue, relative to native lysozyme. This suggests that the increased activity was due to an increased kcat in the catalysis reaction. The H-D exchange rate of the N-1 proton in the Trp63 which is located in the active site cleft, was enhanced in the Del-Arg14His15 lysozyme, while 2-D proton NMR analysis revealed no conformational change around Trp63. We conclude that some sort of fluctuation at the active site might be required for the manifestation of activity. This theory is supported by the finding that the Del-Arg14His15 lysozyme showed a shift in temperature dependency of activity to lower temperatures compared with that of native lysozyme.
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PMID:Lysozyme requires fluctuation of the active site for the manifestation of activity. 793 4

Quenched-flow hydrogen exchange labeling, monitored by 1H NMR and electrospray ionization mass spectrometry (ESI-MS), has been employed in conjunction with stopped-flow circular dichroism and fluorescence to study the kinetic refolding from guanidinium chloride of a derivative of hen lysozyme in which one of the four disulfide linkages (Cys6-Cys127) has been selectively chemically reduced and carboxymethylated (CM6,127-lysozyme). Removal of this disulfide bridge has little effect on the structure and activity of the native enzyme, and the overall kinetics of refolding are very similar to those of the unmodified protein. A substantial amount of secondary structure is formed within 2 ms of the initiation of folding, followed by the slower formation of tertiary interactions characteristic of the native state, which are attained with a time constant (tau) of ca. 200 ms. There is clear evidence for fast and slow refolding populations, as in the intact protein. Folding of the three-disulfide derivative does, however, exhibit a major difference from that of the intact protein under the same final refolding conditions, in that the transient intermediate on the major refolding pathway of the intact protein, having persistent structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labeling during folding of the three-disulfide derivative. This suggests that the disulfide bond linking the N- and C-terminal regions of the protein is crucial for stabilization of the partially folded intermediate. In addition, the overshoot in the far-UV CD and the fluorescence minimum, both of which are attributed to non-native interactions, is not observed in the folding of CM6,127-lysozyme. That the lack of a detectable stable intermediate in the folding of CM6,127-lysozyme does not significantly affect the rate of attainment of the native state of the protein supports the proposed independent nature of the two folding domains and, as the Cys6-Cys127 disulfide bond is located in the alpha-domain, indicates that the rate-limiting step in folding of the intact protein, as well as of the three-disulfide derivative, involves stabilization of the beta-domain. The role of disulfide bridges in the formation and maintenance of the three-dimensional fold of proteins and in facilitating the observation of marginally stable intermediate species is discussed.
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PMID:Kinetic consequences of the removal of a disulfide bridge on the folding of hen lysozyme. 794 9

The complete unfolding of a protein involves the disruption of non-covalent intramolecular interactions within the protein and the subsequent hydration of the backbone and amino acid side-chains. The magnitude of the thermodynamic parameters associated with this process is known accurately for a growing number of globular proteins for which high-resolution structures are also available. The existence of this database of structural and thermodynamic information has facilitated the development of statistical procedures aimed at quantifying the relationships existing between protein structure and the thermodynamic parameters of folding/unfolding. Under some conditions proteins do not unfold completely, giving rise to states (commonly known as molten globules) in which the molecule retains some secondary structure and remains in a compact configuration after denaturation. This phenomenon is reflected in the thermodynamics of the process. Depending on the nature of the residual structure that exists after denaturation, the observed enthalpy, entropy and heat capacity changes will deviate in a particular and predictable way from the values expected for complete unfolding. For several proteins, these deviations have been shown to exhibit similar characteristics, suggesting that their equilibrium folding intermediates exhibit some common structural features. Employing empirically derived structure-energetic relationships, it is possible to identify in the native structure of the protein those regions with the higher probability of being structured in equilibrium partly folded states. In this work, a thermodynamic search algorithm aimed at identifying the structural determinants of the molten globule state has been applied to six globular proteins; alpha-lactalbumin, barnase, IIIGlc, interleukin-1 beta, phage T4 lysozyme and phage 434 repressor. Remarkably, the structural features of the predicted equilibrium intermediates coincide to a large extent with the known structural features of the corresponding intermediates determined by NMR hydrogen-exchange experiments.
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PMID:Structure based prediction of protein folding intermediates. 807 72

One to four alanines were inserted by site-directed mutagenesis at three different locations within the alpha-helix comprising residues 39 to 50 in bacteriophage T4 lysozyme. All insertion mutants were correctly folded and catalytically active although the insertions led to a thermal destabilization by 1.1 to 4.2 kcal/mol when compared to wild-type. Variants that restored part of the loss in stability associated with the initial alanine insertions could be found by randomizing the inserted amino acids. In selected cases, directed mutagenesis of adjacent residues was also used to regain stability. Structural information obtained from X-ray crystallography and/or 2D-NMR for 10 different variants showed two distinct ways in which the protein responded to the amino acid insertions: (1) The inserted amino acids were incorporated into the helix by replacing preceding wild-type amino acids and causing a shift in register towards the N terminus. As a consequence, wild-type amino acids were translocated from the helix into the preceding loop. (2) Insertions caused a "looping out" within the alpha-helix. In this case the perturbation was confined to a minimal region in the immediate vicinity of the insertion. No change in the length of the helix was detected in either case. The structural response appears to be determined by the maintenance of the hydrophobic interface between the helix and the rest of the protein. This interface remains essentially intact in all variant structures. The results exemplify the plasticity and the adaptability of the protein structure which allows the incorporation of additional amino acids into a secondary structure element without large structural perturbations, as long as vital internal interactions are preserved. They also suggest that loops in proteins related by evolution can vary in length not only because of insertions within the loops themselves but also as a consequence of insertions within neighboring secondary structure elements.
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PMID:Accommodation of amino acid insertions in an alpha-helix of T4 lysozyme. Structural and thermodynamic analysis. 811

In order to elucidate the role of the aromatic ring in recognition of the sugar ring, Trp-62 of hen egg white lysozyme, which is proposed on the basis of x-ray crystallography data to make contact with a sugar ring through van der Waals interaction, was replaced with aliphatic amino acids (Leu, Ile, Val, and Ala) and Gly by site-directed mutagenesis. In spite of the loss of the aromatic effect, these mutant lysozymes, except for the Trp-62-->Gly mutant, showed higher bacteriolytic activity than the wild-type lysozyme. Furthermore, the Trp-62-->Gly mutant still retained appreciable bacteriolytic activity. On the other hand, by these replacements, the enzymatic activities toward non-charged substrates were markedly reduced. Additionally, the side-chain structure of position 62 was found to be largely responsible for recognition of a saccharide ring in its active site cleft. NMR analysis of the Trp-62-->Leu and Trp-62-->Gly mutants indicated that the structural effects of Trp-62 replacements were localized in the loop region around position 62 and the part of the beta-sheet containing the hydrogen bonding network important for enzymatic activity. Thus, we conclude that Trp-62 not only interacts with oligosaccharide through van der Waals contact, but also maintains the local structural conformation to produce the lysozyme-oligosaccharide interaction.
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PMID:Functional and structural role of a tryptophan generally observed in protein-carbohydrate interaction. TRP-62 of hen egg white lysozyme. 812 14

The cell adhesive protein RGD8 has been constructed using a yeast expression system by inserting eight amino acid residues (TGRGDSPA) between Val74 and Asn75 of human lysozyme [Yamada et al. (1993) J. Biol. Chem. 268, 10588-10592]. Purified RGD8 from yeast culture supernatant was found to contain glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the inserted Thr residue in the RGD8 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of four or five hexose residues in the glycosylated variants. Only mannose was detected in the sugar analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants, and the structures of these carbohydrate chains were identified as Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha and Man alpha 1-3Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha by 1H-NMR spectroscopy. No other glycosylation was found, although the RGD8 molecule possesses a total of 13 Thr and Ser residues. In addition, no O-glycosylation was observed when the RGD8 protein was expressed in mouse L-cells. Thus, this O-glycosylation looks specific for yeast and the site of the Thr residue. The O-glycosylated variants of RGD8 exhibited a high level of adhesion activity to baby hamster kidney cells, which was almost comparable to that of the unglycosylated form.
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PMID:Site-specific O-glycosylation of cell adhesive lysozyme in yeast. 814 92


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