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)

Jones et al. have reported that the epsilon-carbons of methionine residues in myoglobin can be enriched with stable isotope (13C) in two steps, i.e., methylation of methionine residues with 13CH3I in the protein and thiolysis using dithiothreitol [Jones, W.C., Rothgeb, T.M., and Gurd, F.R.N. (1976) J. Biol. Chem. 251,7452-7460]. Using their method, we failed to prepare active lysozyme in which the epsilon-carbons of methionine residues are enriched with 13C, because many side reactions took place under the thiolysis condition (pH 10.5, 37 degrees C). When we employed 2-aminothiophenol as a reagent for thiolysis, the reduction proceeded under a weakly acidic condition to afford fully active lysozyme, in which the epsilon-carbons of two methionine residues were enriched with 13C, in a 30% yield. Analysis of the 13C-edited NOESY spectra of 13C-enriched methionine lysozyme in the absence and presence of a substrate analogue indicated the occurrence of conformational change around Met 105 in lysozyme.
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PMID:An improved method for preparing lysozyme with chemically 13C-enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis. 949 59

When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)-lysozyme). The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid. A Met(-2)Pro(-1) or Met(-2)Ser(-1) sequence was introduced at the N-terminus of hen egg-white lysozyme. The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine. From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(-1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N-terminus of hen egg-white lysozyme (Pro(-1)-lysozyme). Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg-white lysozyme.
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PMID:Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser. 951 23

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts < or = 0.3 A); in other cases mainchain atoms moved up to 2 A. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) A(-3). Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu --> Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).
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PMID:The response of T4 lysozyme to large-to-small substitutions within the core and its relation to the hydrophobic effect. 951 71

Using hen lysozyme in which the epsilon-carbons of two methionine residues are enriched with 13C nuclei, we found that there is a subtle difference in the chemical shift of the epsilon-carbon resonances between Met 12 and Met 105 in thermally denatured lysozyme without any reduction of disulfide bonds at pD 3.8, and also in reduced S-alkylated lysozyme at pD 3.8 and 35 degrees C. The difference in the chemical shift was abolished on digestion with TPCK-trypsin and the chemical shifts of both resonances converged to that of Met 12, whose chemical shift is identical to that in the randomly coiled state. Therefore, it is suggested that the chemical shift in the epsilon-carbon resonance of Met 105 is different from that in the randomly coiled state due to an interaction involving Met 105. In order to locate the interaction involving Met 105, fragmentation of the reduced S-alkylated lysozyme into the peptides was carried out by means of chemical cleavage or specific endoprotease digestion. As a result, the local interaction of Met 105 or the residues around Met 105 with eleven residues at the C-terminus of lysozyme is suggested to occur.
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PMID:Detection of a local interaction of hen lysozyme under highly denaturing conditions using chemically 13C-enriched methionine resonance. 953 8

The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met --> Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.
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PMID:Context-dependent protein stabilization by methionine-to-leucine substitution shown in T4 lysozyme. 954 9

An aqueous fraction (10-300 micrograms/mL) of the ethanol extract of the leaves of Cissampelos sympodialis Eichl inhibited N-formyl-Met-Leu-Phe (fMLP)-induced release of lysozyme and myeloperoxidase from human neutrophils. Inhibition by the fraction, as well as by dibutyryl-cAMP and prostaglandin E2, was substantially greater when the cells were pretreated with the phosphodiesterase (PDE) inhibitor isobutyl methyl xanthine (IBMX) indicating that the effect may be mediated by cAMP. Measurement of intracellular cAMP levels showed that the fraction (30-100 micrograms/mL) increased the nucleotide levels in IBMX-pretreated neutrophils which was unaffected by propranolol. Cyclic AMP dependent protein kinase A activity was also increased by the fraction (1.5-100 micrograms/mL). Superoxide anion generation induced by fMLP in cytochalasin B-treated cells primed with PAF was not inhibited by the aqueous fraction. The results indicate that the aqueous fraction of Cissampelos sympodialis inhibits neutrophil degranulation by a cAMP-dependent mechanism which may be relevant to the use of the plant as an anti-asthmatic agent in folk medicine.
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PMID:Effects of the aqueous fraction of the ethanol extract of the leaves of Cissampelos sympodialis Eichl. in human neutrophils. 1018 43

The formyltetrapeptides for-Met-Leu-Leu-Phe-OMe 1, for-Met-Leu-Aib-Phe-OMe 2, for-Met-Leu-Ac6c-Phe-OMe 3, for-Met-Leu-Pro-Phe-OMe 4, for-Met-Pro-Pro-Phe-OMe 5, for-Met-Aib-Aib-Phe-OMe 6, for-Met-Pro-Aib-Phe-OMe 7 and for-Met-Aib-Pro-Phe-OMe 8 were synthesized and biologically tested on human neutrophils in an attempt to evaluate the specific receptor pocket dimensions and features. Our results indicate that the shift in the Phe residue to the fourth position in these compounds strongly reduces chemotactic response, but is efficacious in triggering superoxide anion production and lysozyme release (order of potency 3 > 2 > 1 > 4 > 6 > 8 > 5 > 7). The potency of the two latter responses correlates well with the affinity data obtained in binding experiments.
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PMID:An evaluation of fMLP pocket dimensions and features using formyltetrapeptides. 1053 39

In an attempt to identify a systematic relation between the structure of a protein and its folding kinetics, the rate of folding was determined for 20 mutants of T4 lysozyme in which a bulky, buried, nonpolar wild-type residue (Leu, Ile, Phe, Val, or Met) was substituted with alanine. Methionine, which approximated the size of the original side chain but which is of different shape and flexibility, was also substituted at most of the same sites. Mutations that substantially destabilize the protein and are located in the carboxy-terminal domain generally slow the rate of folding. Destabilizing mutations in the amino-terminal domain, however, have little effect on the rate of folding. Mutations that have little effect on stability tend to have little effect on the rate, no matter where they are located. These results suggest that, at the rate-limiting step, elements of structure in the C-terminal domain are formed and have a structure similar to that of the fully folded protein. Consistent with this, two variants that somewhat increase the rate of folding (Phe104 --> Met and Val149 --> Met) are located within the carboxy-terminal domain and maintain or improve packing with very little perturbation of the wild-type structure.
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PMID:Methionine and alanine substitutions show that the formation of wild-type-like structure in the carboxy-terminal domain of T4 lysozyme is a rate-limiting step in folding. 1054 67

To minutely understand the effect of foreign N-terminal residues on the conformational stability of human lysozyme, five mutant proteins were constructed: two had Met or Ala in place of the N-terminal Lys residue (K1M and K1A, respectively), and others had one additional residue, Met, Gly or Pro, to the N-terminal Lys residue (Met(-1), Gly(-1) and Pro(-1), respectively). The thermodynamic parameters for denaturation of these mutant proteins were examined by differential scanning calorimetry and were compared with that of the wild-type protein. Three mutants with the extra residue were significantly destabilized: the changes in unfolding Gibbs energy (DeltaDeltaG) were -9.1 to -12.2 kJ.mol-1. However, the stability of two single substitutions at the N-terminal slightly decreased; the DeltaDeltaG values were only -0.5 to -2.5 kJ.mol-1. The results indicate that human lysozyme is destabilized by an expanded N-terminal residue. The crystal structural analyses of K1M, K1A and Gly(-1) revealed that the introduction of a residue at the N-terminal of human lysozyme caused the destruction of hydrogen bond networks with ordered water molecules, resulting in the destabilization of the protein.
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PMID:Effect of foreign N-terminal residues on the conformational stability of human lysozyme. 1056 12

In order to try to better understand the role played by strain in the structure and stability of a protein a series of "small-to-large" mutations was made within the core of T4 lysozyme. Three different alanine residues, one involved in backbone contacts, one in side-chain contacts, and the third adjacent to a small cavity, were each replaced with subsets of the larger residues, Val, Leu, Ile, Met, Phe and Trp. As expected, the protein is progressively destabilized as the size of the introduced side-chain becomes larger. There does, however, seem to be a limit to the destabilization, suggesting that a protein of a given size may be capable of maintaining only a certain amount of strain. The changes in stability vary greatly from site to site. Substitution of larger residues for both Ala42 and Ala98 substantially destabilize the protein, even though the primary contacts in one case are predominantly with side-chain atoms and in the other with backbone. The results suggest that it is neither practical nor meaningful to try to separate the effects of introduced strain on side-chains from the effects on the backbone. Substitutions at Ala129 are much less destabilizing than at sites 42 or 98. This is most easily understood in terms of the pre-existing cavity, which provides partial space to accommodate the introduced side-chains. Crystal structures were obtained for a number of the mutants. These show that the changes in structure to accommodate the introduced side-chains usually consist of essentially rigid-body displacements of groups of linked atoms, achieved through relatively small changes in torsion angles. On rare occasions, a side-chain close to the site of substitution may change to a different rotamer. When such rotomer changes occur, they permit the structure to dissipate strain by a response that is plastic rather than elastic. In one case, a surface loop moves 1.2 A, not in direct response to a mutation, but in an interaction mediated via an intermolecular contact. It illustrates how the structure of a protein can be modified by crystal contacts.
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PMID:The introduction of strain and its effects on the structure and stability of T4 lysozyme. 1062 13


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