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)

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.
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PMID: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. 754 86

Vibrational circular dichroism (VCD) has been shown to be sensitive to secondary structure in proteins and peptides and has been used as the basis for quantitative secondary-structure-prediction algorithms. However, the accuracy of these algorithms is not matched by the apparent qualitative sensitivity of the VCD spectra. This report provides examples of the use of VCD to follow structural change spectrally and to clarify the qualitative nature of the structural changes underlying the spectral variation. The VCD spectra and the complementary UV electronic CD (ECD) and FTIR spectra of alpha-lactalbumin (LA) have been studied as a function of pH, denaturation, Ca2+ ion and solvent conditions for several species. Spectral data for lysozyme were compared with those of LA because of their very similar crystal structures. In fact, these proteins in D2O-based pH 7 solution have quite different spectra using these optical techniques. Even for the LA proteins, the human differs from the bovine and goat species. Furthermore, under low pH conditions, where the LAs are in a reversibly denatured, molten globule form, the spectra are more similar, species variation is minimal and the spectral differences from lysozyme are in fact smaller. Our data are consistent with native, pH 7, alpha-lactalbumin having a less well organized structure than lysozyme, possibly in a dynamic sense. Conversely, in the low-pH, molten globule form of LA, tertiary structure is lost which could relax constraints that might distort the helical segments in the native form. The differences between the interpretation of our results and those from X-ray and NMR data may be due to motional sampling of various geometries in LA which all contribute to the spectral signatures seen in optical spectra but whose contributions are washed out in NMR or frozen out in the crystal structure. Part of this flexibility may relate to the rather large 3(10)-helical content in the LA protein structure. Fluctionality may have specific functional effects, perhaps allowing LA to bind better to beta-galactosyl transferase and form the biologically active lactose synthetase complex.
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PMID:Empirical studies of protein secondary structure by vibrational circular dichroism and related techniques. Alpha-lactalbumin and lysozyme as examples. 754 41

The chemical modification of Asp101 which is located at the upper end-most site (site A) of the binding cleft of hen egg white lysozyme affects the sugar residue binding of the midmost site (site C) in addition to that of site A, and results in the considerable decrease in the enzymic activity [Fukamizo, T., Hayashi, K. & Goto, S. (1986) Eur. J. Biochem. 158, 463-467]. In the present study, Asp101 was modified with histamine and converted to [2-imidazol-4(5)-ylethyl]asparagine. Contrary to the findings described above, the specific activity of the modified lysozyme was higher than that of the native lysozyme by a factor of about two, and the loss of sugar residue binding ability caused by the modification was found to be restricted to site A. From the H-NMR spectra of the modified lysozyme, the pKa value of the imidazolylethyl group covalently attached to Asp101 was 7.1, and was higher than that of N-acetylhistidinemethylamide (6.65). This indicates that the imidazolylethyl moiety is not exposed to the solvent but adheres to the surface of the lysozyme molecule in an unidentified manner. When N-acetylglucosamine trisaccharide [GlcNAc)3] was added to the modified lysozyme, the 1H-NMR signals of H2 and H4 of the imidazolylethyl group were strongly affected. This indicates that the imidazolylethyl moiety is located near (GlcNAc)3 binding region. When the H gamma signal of Ile98 was saturated, nuclear Overhauser effects were observed on H2 and H4 resonances of the imidazolylethyl moiety. NOE was also observed on the signal of Trp63 H6 upon the saturation of the H4 signal of the imidazolylethyl moiety. Thus, the imidazolylethyl moiety should be located near Trp63 and Ile98, which are in the hydrophobic box most proximal to the sugar binding cleft. This situation of the imidazolylethyl moiety did not result in steric hindrance to the sugar residue binding at sites B and C. The modification affected only the sugar residue binding at site A, and resulted in the enhanced activity.
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PMID:Hen-egg-white lysozyme modified with histamine. State of the imidazolylethyl group covalently attached to the binding site and its effect on the sugar-binding ability. 762 85

The high-affinity calcium-binding sites of bovine and human alpha-lactalbumin as well as equine lysozyme were analyzed by 113Cd NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched 113Cd2+ results in a signal at delta = -75.9 ppm corresponding to the metal ion bound to the lone Ca(2+)-binding site of the protein. A peak at virtually the identical resonance position (delta = -77.1 ppm) was observed in the analogous experiment with bovine alpha-lactalbumin. In addition, a signal upfield of these (delta = -94.7 ppm) was observed for 113Cd(2+)-substituted human alpha-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca(2+)-binding proteins. The field dependence of the 113Cd signals for all three proteins and bovine calmodulin were compared. At each field, the 113Cd signal linewidths for the alpha-lactalbumins and the lysozyme are somewhat broader than those observed for the EF-hand protein. In addition, the 113Cd linewidths for the lactalbumins and the lysozyme, especially bovine alpha-lactalbumin, increase dramatically with the square of the magnetic field strength, indicative of the presence of nuclear relaxation via chemical shift anisotropy and chemical exchange. The protein-bound 113Cd signals for the alpha-lactalbumins are also markedly affected by changes in the amount of K+ present, since Cd2+ and K+ can compete for occupation of the high-affinity Ca(2+)-site. Their linewidths also to some extent depend on the concentration of the protein itself.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cadmium-113 NMR studies of bovine and human alpha-lactalbumin and equine lysozyme. 762 32

Solid phase methods have been used to synthesise a peptide corresponding to residues 38-51 of T4 lysozyme. The peptide, LYS(38-51), encompasses helix B in the crystal structure of T4 lysozyme. CD and 1H-NMR analysis showed that the peptide was unstructured in aqueous solution but adopted a helical conformation in the more hydrophobic environment provided by 50% TFE and SDS micelles. The solution structure derived from the NMR data was similar to that of the helix in the X-ray structure, although there was some fraying at the N-terminus.
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PMID:CD and NMR determination of the solution structure of a peptide corresponding to T4 lysozyme residues 38-51. 763 21

Three 1000 ps molecular dynamics simulations of hen lysozyme have been compared with a range of experimental NMR parameters in order to gain insight into the dynamical properties of the protein and to assess the significance of the motional events observed in the simulations. The simulations, one in vacuum and two in water, were used to estimate interproton distances (for comparison with NOE data), 3JHN alpha and 3J alpha beta coupling constants and 1H-15N order parameters. Comparison of these values with experimental data, particularly NOEs, enabled force field-induced changes to the structure during the simulations to be recognized. It has been shown, however, that these changes can be largely eliminated by slight modifications to the force field. Using a simulation performed in water with this modified force field, it has been found that 1H-15N order parameters calculated for side chain groups in particular correlate well with experimental values and reflect the substantial dependence of these motional properties on the environment, particularly surface exposure, in which the side chain is found. In this case, the simulation then provides models for the motional processes giving rise to the observed experimental data. The results indicate that the order parameter values reflect primarily the number of torsion angles about which rotameric interchange occurs. In addition to local motions, the two different domains of lysozyme have been found to behave differently in the simulations. Possible implications of these differences for the interpretation of unfolding simulations and experimental observations of folding intermediates for lysozyme are discussed.
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PMID:Comparison of MD simulations and NMR experiments for hen lysozyme. Analysis of local fluctuations, cooperative motions, and global changes. 766 73

A method was developed to evaluate the association constant at physiological pH (pH 7.5) between a lysozyme and the Fab fragment derived from anti-lysozyme monoclonal antibody 37-7, which was immobilized to the adsorbent for HPLC. Comparison of the association constants between lysozymes and the immobilized Fab fragment indicated that mAb 37-7 recognized the prominently exposed regions (hills and ridges) around His15 of hen lysozyme, but His15 itself was not directly involved in the binding with mAb 37-7. Moreover, the epitope was confirmed by the reactivity of His15 with monoiodoacetic acid in the presence of mAb 37-7. The association constant of 15-carboxymethylated histidine lysozyme (15CM lysozyme) with the immobilized Fab fragment was smaller by one-seventh than that of 15-carboxamidated histidine lysozyme, though the side chains introduced were almost identical in size. From the pH titration of 15CM lysozyme with 13C-enriched carboxyl group by use of 13C-NMR, the pKa of the introduced carboxyl group was evaluated to be 5.06. Since the carboxyl group was fully ionized under the conditions of measurement (pH 7.5), electrostatic repulsion was found to disturb severely the association between mAb 37-7 and hen lysozyme. Moreover, it was demonstrated that, because of the high reproducibility of measurement, the immobilized Fab fragment could detect subtle differences in the surface structure of lysozymes.
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PMID:Detection of subtle differences in the surface structure of lysozymes by use of an immobilized Fab fragment. 768 15

15N-labeled hen lysozyme has been studied by 2D and 3D NMR in order to characterize its dynamic behavior. The resonances of all main-chain amide nitrogen atoms were assigned, as were resonances of nitrogen atoms in 28 side chains. Relaxation measurements for the main-chain and arginine and tryptophan side-chain 15N nuclei used standard methods, and those for the 15N nuclei of asparagine and glutamine side chains used pulse sequences designed to remove unwanted relaxation pathways in the NH2 groups. The calculated order parameters (S2) show that the majority of main-chain amides undergo only small amplitude librational motions on a fast time scale (S2 > or = 0.8). Increased main-chain motion (0.5 < S2 < 0.8) is observed for a total of 19 residues located at the C-terminus, in loop and turn regions, and in the first strand of the main beta-sheet. Order parameters derived for the side chains range from 0.05 to 0.9; five of the six tryptophan residues have high order parameters (S2 > or = 0.8), consistent with their location in the closely packed core of the protein, whereas the order parameters between 0.05 and 0.3 for arginine residues confirm increased side-chain mobility at the protein surface. Order parameters for the side chains of asparagine and glutamine residues range from 0.2 to 0.8; high values are found for side chains that have low solvent accessible surfaces and well-defined chi 1 values, as measured by 3J alpha beta coupling constants. Many of the main-chain and side-chain groups with low order parameters have higher than average temperature factors in X-ray crystal structures and increased positional uncertainty in NMR solution structures. They also tend to lack persistent hydrogen bond interactions and protection against amide hydrogen exchange. The most significant correlations are found between residues with low order parameters and high surface accessibility in both crystal and solution structures. The results suggest that a lack of van der Waals contacts is a major determinant of side-chain and main-chain mobility in proteins.
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PMID:Structural determinants of protein dynamics: analysis of 15N NMR relaxation measurements for main-chain and side-chain nuclei of hen egg white lysozyme. 769 70

The binding of N,N',N",N"'-tetraacetylchitotetraitol [(GlcNAc)4-ol] to hen egg white lysozyme was investigated by fluorescence and 1H NMR spectroscopy. From observation of changes in the fluorescence intensity, the association constants of (GlcNAc)4-ol and (GlcNAc)3 were found to be 0.70 x 10(5) and 1.07 x 10(5) M-1, respectively, at pH 5.0 and 30 degrees C. The lack of a substantial difference between the association constants suggests that the binding mode of the (GlcNAc)3 moiety of (GlcNAc)4-ol is basically similar to that of (GlcNAc)3, but that the N-acetylglucosaminitol residue of (GlcNAC)4-ol does not interact significantly with lysozyme. On the other hand, 1H NMR spectroscopy revealed a minor difference in the binding modes of the two saccharides. For most of the 1H signals responding to saccharide binding, such as those of Trp 63 H2, Trp 28 H5, and Ile 98 H gamma 1, the chemical shift changes induced by (GlcNAc)4-ol were almost identical to those induced by (GlcNAc)3. However, the effect of binding on the signals of Asn 59 H alpha and Trp 108 indole N1H, which are located near subsite C, was different for (GlcNAc)4-ol and (GlcNAc)3. Thus it is inferred that the binding mode of the first sugar residue of (GlcNAc)4-ol to subsite C is somewhat different from that of (GlcNAc)3.
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PMID:Binding mode of N,N',N",N"'-tetraacetylchitotetraitol to hen egg white lysozyme. 769 63

The conformation, in solution, of a peptide corresponding to residues 59-81 from T4 lysozyme [LYS(59-81)] has been determined by 1H NMR and CD spectroscopy. This peptide spans the region corresponding to helix C in the crystal structure of T4 lysozyme. Secondary structure predictions indicated that the peptide would possibly be helical in an aqueous environment, but in a more hydrophobic environment the peptide would certainly adopt a helical conformation. This prediction was confirmed by the far-UV CD and NMR studies, which showed the peptide to be relatively unstructured in aqueous solution and significantly helical in the presence of either TFE or SDS micelles, although the 1H NMR results did give some indication of the presence of nascent helix in aqueous solution. For LYS(59-81), in TFE, the three-dimensional structure derived from the NMR data showed that the helix had a more pronounced curvature than the gradual bend observed in the crystal structure.
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PMID:Conformation of a peptide corresponding to T4 lysozyme residues 59-81 by NMR and CD spectroscopy. 772 68


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