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)

Amide exchange kinetics were used to probe the conformation of hen egg-white lysozyme complexed with the anti-lysozyme monoclonal antibody HyHEL-5. Following the technique developed by Paterson et al. [(1990) Science 249, 755-759] we used two-dimensional NMR to measure amide exchange kinetics of the lysozyme amide protons in the lysozyme-antibody complex. A total of 15 amide protons showed altered exchange kinetics in the presence of the complex. Five of these 15 protons reside on residues that are found within the epitope as defined by X-ray crystallography. Five residues are located at the perimeter of the epitope. The remaining five residues are removed from the epitope. The perturbation of amide exchange rates at sites distant from the epitope indicates that the formation of antigen-antibody complexes can produce changes in the antigen at sites that are quite distant from the structural epitope.
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PMID:Long-range changes in a protein antigen due to antigen-antibody interaction. 138 91

We have undertaken a new and more detailed Fourier-transform infrared (FTIR) spectroscopic study of alpha-lactalbumin (in D2O solution) aimed at correlating its secondary structures to observed Amide I' infrared bands. The spectra reported here were interpreted in light of the recently determined crystal structure of alpha-lactalbumin and by comparison with the spectra and structure of the homologous protein lysozyme. Of particular importance is the new evidence supporting the assignment of the band at 1639 cm-1 to 3(10)-helices. This assignment is in excellent agreement with one based on theoretical and experimental studies of 3(10)-helical polypeptides. The frequency observed for 3(10)-helices is distinctly different from that at which alpha-helices are typically found (viz., around 1655 cm-1). In the present study, two bands are clearly resolved in the latter region at 1651 and 1659 cm-1. Both are apparently associated with alpha-helices. These results suggest that for D2O solutions of globular proteins. FTIR spectroscopy can be a facile method for detecting the presence of these two different types of helical conformation and distinguishing between them. This provides a distinct advantage over ultraviolet circular dichroism spectroscopy (UV-CD). This work also provides a basis for future studies of alpha-lactalbumin which examine the effects of environment (e.g., pH, temperature) and ligands (e.g., Ca2+, Mn2+) on its conformation.
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PMID:Infrared spectroscopic discrimination between alpha- and 3(10)-helices in globular proteins. Reexamination of Amide I infrared bands of alpha-lactalbumin and their assignment to secondary structures. 191 8

Amide hydrogen/deuterium exchange behaviour has been studied for all of the peptide amides of hen lysozyme by means of two-dimensional n.m.r. spectroscopy. The amides have been grouped into four categories on the basis of their rates of exchange in solution at pH 4.2 and 7.5. The distribution of the amides into the different categories has been examined in the light of the crystallographic structural information, considering the type of secondary structure, the nature of hydrogen bonding and the distance from the protein surface. None of these features was found to determine uniquely the pattern of hydrogen exchange rates within the protein. The exchange behaviour of the individual amides could, however, in general be rationalized by a combination of these features. Hydrogen exchange was also monitored in both tetragonal and triclinic crystals of lysozyme, by allowing exchange to take place in the crystals prior to dissolution and recording of n.m.r. spectra under conditions where further exchange was minimized. This enabled direct comparison to be made of the exchange behaviour in the crystals and solution. A reduction in exchange rate was observed in the crystalline state relative to solution for a substantial number of amides and distinct differences between exchange in the different crystals could be observed. These differences between the solution and the different crystal states do not, however, correlate in a simple manner with proximity to intermolecular contacts in the crystals. However, the existence of these contacts, which are on the surface of the protein molecule, have a profound effect on the exchange of amides in the interior of the protein. The results indicate that the spectrum of fluctuations giving rise to hydrogen exchange may be significantly altered by the intermolecular interactions present within the crystalline state.
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PMID:A nuclear magnetic resonance study of the hydrogen-exchange behaviour of lysozyme in crystals and solution. 201 Sep 18

Supercritical CO2 was used as an antisolvent to form protein particles that exhibited minimal loss of activity upon reconstitution. Organic protein solutions were sprayed under a variety of operating conditions into the supercritical fluid, causing precipitation of dry, microparticulate (1-5 microns) protein powders. Three proteins were studied: trypsin, lysozyme, and insulin. Amide I band Raman spectra were used to estimate the alpha-helix and beta-sheet structural contents of native and precipitate powders of each protein. Analysis of the Raman spectral revealed minimal (lysozyme), intermediate (trypsin), and appreciable (insulin) changes in secondary structure with respect to the commercial starting materials. The perturbations in secondary structure suggest that the most significant event during supercritical fluid-induced precipitation involved the formation of beta-sheet structures with concomitant decreases of alpha-helix. Amide I band Raman and Fourier-transform infrared (FTIR) spectra indicate that higher operating temperatures and pressures lead to more extensive beta-sheet-mediated intermolecular interactions in the precipitates. Raman and FTIR spectra of redissolved precipitates are similar to those of aqueous commercial proteins, indicating that conformational changes were reversible upon reconstitution. These results suggest that protein precipitation in supercritical fluids can be used to form particles suitable for controlled release, direct aerosol delivery to the lungs, and long-term storage at ambient conditions.
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PMID:Precipitation of proteins in supercritical carbon dioxide. 877 54

Temperature coefficients have been measured by 2D NMR methods for the amide and C alpha H proton chemical shifts in two globular proteins, bovine pancreatic trypsin inhibitor and hen egg-white lysozyme. The temperature-dependent changes in chemical shift are generally linear up to about 15 degrees below the global denaturation temperature, and the derived coefficients span a range of roughly -16 to +2 ppb/K for amide protons and -4 to +3 ppb/K for C alpha H. The temperature coefficients can be rationalized by the assumption that heating causes increases in thermal motion in the protein. Precise calculations of temperature coefficients derived from protein coordinates are not possible, since chemical shifts are sensitive to small changes in atomic coordinates. Amide temperature coefficients correlate well with the location of hydrogen bonds as determined by crystallography. It is concluded that a combined use of both temperature coefficients and exchange rates produces a far more reliable indicator of hydrogen bonding than either alone. If an amide proton exchanges slowly and has a temperature coefficient more positive than -4.5 ppb/K, it is hydrogen bonded, while if it exchanges rapidly and has a temperature coefficient more negative than -4.5 ppb/K, it is not hydrogen bonded. The previously observed unreliability of temperature coefficients as measures of hydrogen bonding in peptides may arise from losses of peptide secondary structure on heating.
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PMID:Temperature dependence of 1H chemical shifts in proteins. 925 42

Synthesis of two chimeric peptides composed of tuftsin and thymic humoral factor-gamma 2 (THF-gamma 2) conjugates was accomplished. Our goal was the generation of novel immunomodulators. Initially, we demonstrate an IL-6 inducing activity of the phagocytic cells stimulant, tuftsin, on murine macrophages. This activity was documented only in the presence of antigen, either KLH or lysozyme. The augmentation was dose dependent, with optimal activity at a concentration of 200 and 20 nM, respectively. The chimeric peptides, either H2N-tuftsin-THF-gamma 2-OH or H2N-THF-gamma 2-tuftsin-OH, were also evaluated in the IL-6 system in the presence of the more potent antigen, KLH. The IL-6 inducing effect was maintained, although maximal activity appeared only at a concentration an order of magnitude greater than that of tuftsin. The chimeric peptides were further tested in an assay evaluating enhancement in murine bone marrow myeloid colony formation, a system in which THF-gamma 2, a T cell stimulant, has an established beneficial effect. The compounds were found to be inactive at the 25-200 ng/ml (14-112 nM) concentration range evaluated. Finally, the chimeric peptides were tested in a combined macrophages-T cells assay, i.e. antigen presentation, in which H2N-tuftsin-THF-gamma 2-OH was found to be more active than either parent peptide, thus representing a possible therapeutic agent.
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PMID:Tuftsin-THF-gamma 2 chimeric peptides: potential novel immunomodulators. 928 43

At high (> 3.5 kbar) pressures and low (< -10 degrees C) temperatures, hen egg-white lysozyme denatures readily and reversibly. Amide hydrogen exchange methods were used to investigate the structure of the pressure-assisted cold-denatured state of lysozyme. Protection factors were obtained for 52 backbone amide protons. The extent of protection of many of these protons is markedly different from that in lysozyme denatured by high temperature, high urea concentration, or chemical modification; specifically, the protection factors are higher and are strongly correlated with elements of secondary structure present in the native state. Furthermore, the pattern of protection factors is similar to that observed in lysozyme during refolding from highly denatured states, particularly during the early stages (< 3.5 ms) of refolding [Gladwin, S. T., & Evans, P. A. (1996) Folding Des. 1, 407]. Previous data on cold-denatured ribonuclease A were reevaluated and compared to known folding intermediates [Houry, W. A. & Scheraga, H. A. (1996) Biochemistry 35, 11734; Udgaonkar, J. B., & Baldwin, R. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8197] to further test the supposition that the pressure-assisted cold-denatured states of proteins resemble the early folding stages.
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PMID:Structure of pressure-assisted cold denatured lysozyme and comparison with lysozyme folding intermediates. 939 55

Lysozyme distribution and conformation in poly(lactic-co-glycolic acid)(PLGA) microspheres was determined using various infrared spectroscopic techniques. Infrared microscopy and confocal laser scanning microscopy indicated that the protein was homogeneously distributed inside the microspheres in small cavities resulting from the water-in-oil emulsification step. Part of the protein was observed at or near the cavity walls, while the rest was located within these cavities. Attenuated total reflectance (ATR) and photoacoustic spectroscopy (PAS) also showed that there is hardly any protein at the surface of the microspheres. Since this microsphere formulation gave a large burst release (ca. 50%), this burst release can not be caused by protein at the surface of the particles. Probably, the protein is rapidly released through pores in the PLGA matrix. Conformational analysis of lysozyme in the PLGA microspheres by KBr pellet transmission suffered from band shape distortion and baseline slope. Despite incomplete subtraction of the PLGA background, a characteristic band of non-covalent aggregates at 1625 cm(-1) was observed in the second derivative spectrum of the protein Amide I region. The other Fourier-transform infrared (FTIR) methods yielded similar results, indicating that the sample preparation procedure did not introduce artifacts. The observed aggregation signal may correspond to the protein adsorbed to the cavity walls inside the microspheres.
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PMID:Lysozyme distribution and conformation in a biodegradable polymer matrix as determined by FTIR techniques. 1088 77

The immobilization of protein molecules on self-assembled monolayers (SAM) by physical interactions and chemical bonding has been studied using atomic force microscopy (AFM). The proteins used for our investigation are bovine serum albumin (BSA), lysozyme (LYZ), and normal rabbit immunoglobulin G (IgG). The surfaces are methyl-, hydroxyl-, carboxylic acid- and aldehyde-terminated SAMs. We found that BSA and LYZ can be readily immobilized on SAMs at their isoelectric point (IEP). The detailed surface morphology of adsorbed proteins varies with the functionality of the SAMs. The strong hydrophobic interaction at the IEP is attributed to immobilization. If the solution pH is deviated from the IEP, proteins may be attached onto the surface via electrostatic interactions. Covalent binding between the aldehyde-terminated SAM and the H2N-groups in the protein results in immobilization of all three proteins. The individual proteins and their orientations on SAMs are clearly resolved from high-resolution AFM images. The stability and bioactivity of these immobilized proteins are also studied.
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PMID:Immobilization of proteins on self-assembled monolayers. 1114 64

Equine lysozyme is a calcium-binding lysozyme and an evolutional intermediate between non-calcium binding c-type lysozyme and alpha-lactalbumin. We constructed a chimeric protein by substituting the fluctuating loop of bovine alpha-lactalbumin with the D-helix of equine lysozyme. The substitution affects the protection factors not only in the fluctuating loop but also in the antiparallel beta-sheet, the A- and B-helices, and the loop between the B-helix and the beta-sheet. Amide protons in these regions of the chimera are more protected from exchange than are those of bovine alpha-lactalbumin. We used model-free analysis based on 15N nuclear magnetic resonance relaxation measurements to investigate the dynamics of the main chain of the chimera and showed that the fluctuating loop of the chimera is as rigid as three major helices. When we analyzed the chemical shift deviations and backbone HN-H(alpha) scalar coupling constants, we found that the chimera showed an alpha-helical tendency in residues around the fluctuating loop. Our results suggest that the replacement of a highly fluctuating loop in a protein with a rigid structural element in a homologous one may be useful to stabilize the protein structure.
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PMID:Stabilization of protein by replacement of a fluctuating loop: structural analysis of a chimera of bovine alpha-lactalbumin and equine lysozyme. 1242 44


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