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The Journal of Experimental Medicine Nov 1966Markedly increased quantities of lysozyme have been found in the serum and urine (ranging to 2.6 g per day) of ten consecutive cases of monocytic and monomyelocytic...
Markedly increased quantities of lysozyme have been found in the serum and urine (ranging to 2.6 g per day) of ten consecutive cases of monocytic and monomyelocytic leukemia. The enzyme has been isolated from the urine of several cases and physicochemically and immunochemically characterized. It is apparently identical to the lysozyme of normal tears, saliva, leukocytes, and serum, but structurally different from the lysozyme of hen's egg white. The activity of the human enzyme assayed with M. lysodeikticus organisms is 3 to 12 times greater than egg white lysozyme at equivalent concentrations. An agar plate method has been developed for quantitating lysozyme activity in small samples (approximately 25 microl) of serum, urine, or other biological fluids. The range and reproducibility of this method were found to be superior to previously available lysozyme assay procedures. Present evidence indicates that lysozyme is the principal, if not the sole, product of the proliferating monocytes in monocytic and monomyelocytic leukemia, and quantitation of serum and urine lysozyme should be a useful diagnostic procedure for these leukemias.
Topics: Aged; Chromatography; Electrophoresis; Female; Humans; Leukemia, Myeloid; Male; Middle Aged; Muramidase; Precipitin Tests; Proteinuria
PubMed: 5224561
DOI: 10.1084/jem.124.5.921 -
PloS One 2021Static light scattering is a popular physical chemistry technique that enables calculation of physical attributes such as the radius of gyration and the second virial...
Static light scattering is a popular physical chemistry technique that enables calculation of physical attributes such as the radius of gyration and the second virial coefficient for a macromolecule (e.g., a polymer or a protein) in solution. The second virial coefficient is a physical quantity that characterizes the magnitude and sign of pairwise interactions between particles, and hence is related to aggregation propensity, a property of considerable scientific and practical interest. Estimating the second virial coefficient from experimental data is challenging due both to the degree of precision required and the complexity of the error structure involved. In contrast to conventional approaches based on heuristic ordinary least squares estimates, Bayesian inference for the second virial coefficient allows explicit modeling of error processes, incorporation of prior information, and the ability to directly test competing physical models. Here, we introduce a fully Bayesian model for static light scattering experiments on small-particle systems, with joint inference for concentration, index of refraction, oligomer size, and the second virial coefficient. We apply our proposed model to study the aggregation behavior of hen egg-white lysozyme and human γS-crystallin using in-house experimental data. Based on these observations, we also perform a simulation study on the primary drivers of uncertainty in this family of experiments, showing in particular the potential for improved monitoring and control of concentration to aid inference.
Topics: Animals; Bayes Theorem; Chickens; Dynamic Light Scattering; Humans; Hydrogen-Ion Concentration; Models, Molecular; Muramidase; Protein Aggregates; Sodium Chloride; gamma-Crystallins
PubMed: 34648536
DOI: 10.1371/journal.pone.0258429 -
PloS One 2016Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6...
Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as well as hydrogen bonding network between (NAG)3 and proteins were mostly conserved. LYZL5 in buffalo and other mammalian species contained additional 10-12 amino acid sequence at c-terminal that matched with ankyrin repeat domain-containing protein 27. Phylogenetic analysis indicated LYZL2 to be most ancient among all the LYZL proteins and that the evolution of LYZL proteins occurred through several gene duplications preceding the speciation of mammals from other vertebrates as distant as reptiles and amphibians.
Topics: Amino Acid Sequence; Animals; Binding Sites; Buffaloes; Catalytic Domain; Cloning, Molecular; Fertility; Male; Models, Molecular; Muramidase; Phylogeny; Protein Conformation; Seminal Plasma Proteins; Sequence Alignment
PubMed: 27832206
DOI: 10.1371/journal.pone.0166321 -
Scientific Reports Feb 2016Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular...
Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in (1)H, (15)N, and (13)C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins.
Topics: Binding Sites; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Dynamics Simulation; Muramidase; Oxygen; Protein Binding; Proteins
PubMed: 26830762
DOI: 10.1038/srep20534 -
Biointerphases Jun 2020Amyloid fibrillation is known to contribute in a variety of diseases including neurodegenerative disorders (e.g., Alzheimer's and Parkinson's disease) and type II...
Amyloid fibrillation is known to contribute in a variety of diseases including neurodegenerative disorders (e.g., Alzheimer's and Parkinson's disease) and type II diabetes. The inhibition of fibrillation has been suggested as a possible therapeutic strategy to prevent neuronal and pancreatic β-cell death associated with amyloid diseases. To this end, strong hydrophobic and π-π interactions between proteins and nanomaterials at the nanobio interface could be used to mitigate the stacking of amyloid structures associated with fibrillation. In this study, the authors show that exfoliated graphene effectively inhibits the formation of amyloid fibrils using a model amyloid-forming protein, viz., hen egg white lysozyme (HEWL). While previous theoretical models posit that hydrophobic and π-π stacking interactions result in strong interactions between graphene and proteins, the authors experimentally identified the presence of additional interfacial charge transfer interactions between HEWL and graphene using micro-Raman spectroscopy and Kelvin probe force microscopy. Their photoluminescence spectroscopy and transmission electron microscopy studies evince that the interfacial charge transfer combined with hydrophobic and π-π stacking interactions, specifically between the nanomaterial and the amino acid tryptophan, increase HEWL adsorption on graphene and thereby inhibit amyloid fibrillation.
Topics: Amyloid; Animals; Benzothiazoles; Chickens; Fluorescence; Graphite; Muramidase; Protein Structure, Secondary; Spectrum Analysis, Raman
PubMed: 32493017
DOI: 10.1116/6.0000019 -
Antimicrobial Agents and Chemotherapy May 2022Enterococcus faecalis, a leading cause of health care-associated infections, forms biofilms and is resistant to many antimicrobial agents. Planktonic-phase E. faecalis...
Enterococcus faecalis, a leading cause of health care-associated infections, forms biofilms and is resistant to many antimicrobial agents. Planktonic-phase E. faecalis is resistant to high concentrations of the enzyme lysozyme, which catalyzes the hydrolysis of -acetylmuramic acid and -acetylglucosamine linkages in peptidoglycan and is also a cationic antimicrobial peptide (CAMP). E. faecalis lysozyme resistance in planktonic cells is stimulated upon activation of the extracytoplasmic function sigma factor SigV via cleavage of the anti-sigma factor RsiV by the transmembrane protease Eep. Planktonically grown E. faecalis lacking is more sensitive than wild-type strains to growth inhibition by lysozyme. This study was initiated to determine whether E. faecalis OG1RFΔ biofilms would be protected from lysozyme. Serendipitously, we discovered that exposure of both E. faecalis OG1RF and OG1RFΔ biofilms to chicken egg white lysozyme resulted in decreases in biofilm cell viability of 3.7 and 3.8 log CFU/mL, respectively. Treatment of biofilms of both strains with recombinant purified human lysozyme was associated with reductions in cell viability of >99.9% for both strains. Lysozyme-treated OG1RF and OG1RFΔ biofilms contained a higher percentage of dead cells by Live/Dead staining and were associated with more extracellular DNA. Heat-inactivated human lysozyme, which was devoid of muramidase activity, as well as the lysozyme-derived CAMP LP9 and the CAMP polymyxin B, decreased biofilm cell viability. These results are consistent with a model in which the CAMP activity, rather than the muramidase activity, of lysozyme causes lysis of E. faecalis biofilm cells despite them having an intact lysozyme resistance-inducing signaling pathway. Finally, lysozyme was also effective in reducing viable biofilm cells of several other E. faecalis strains, including the vancomycin-resistant strain V583 and multidrug-resistant strain MMH594. This study demonstrates the potential for lysozyme to be developed as a novel antibiofilm therapeutic.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antimicrobial Peptides; Biofilms; Enterococcus faecalis; Muramidase; Plankton
PubMed: 35446133
DOI: 10.1128/aac.02339-21 -
Cellular and Molecular Life Sciences :... Mar 2004In amyloid related diseases, proteins form fibrillar aggregates with highly ordered beta-sheet structure regardless of their native conformations. Formation of such... (Review)
Review
In amyloid related diseases, proteins form fibrillar aggregates with highly ordered beta-sheet structure regardless of their native conformations. Formation of such amyloid fibrils can be reproducible in vitro using isolated proteins/peptides, suggesting that amyloid fibril formation takes place as a result of protein conformational change. In vitro studies revealed that perturbation of the native structure is important for the fibril formation, and it is suggested that the mechanisms of amyloid fibril formation share the mechanisms of protein folding. In particular, amyloid fibril formation is similar to one of the common features of proteins, i.e. amorphous aggregation upon partial unfolding, which is likely driven by hydrophobic interactions through exposed protein interior. However, these molecular associations are distinct phenomena, and identifying factors that lead to amyloid fibril formation would precede our understanding of the mechanisms of amyloid fibrillization. The necessity of understanding the nature of protein denatured states is also suggested.
Topics: Amyloid; Humans; Muramidase; Mutation; Prions; Protein Folding; beta 2-Microglobulin
PubMed: 15004691
DOI: 10.1007/s00018-003-3264-8 -
Proteins Oct 2020A fully atomistic (AT) modeling of biological macromolecules at relevant length- and time-scales is often cumbersome or not even desirable, both in terms of...
A fully atomistic (AT) modeling of biological macromolecules at relevant length- and time-scales is often cumbersome or not even desirable, both in terms of computational effort required and a posteriori analysis. This difficulty can be overcome with the use of multiresolution models, in which different regions of the same system are concurrently described at different levels of detail. In enzymes, computationally expensive AT detail is crucial in the modeling of the active site in order to capture, for example, the chemically subtle process of ligand binding. In contrast, important yet more collective properties of the remainder of the protein can be reproduced with a coarser description. In the present work, we demonstrate the effectiveness of this approach through the calculation of the binding free energy of hen egg white lysozyme with the inhibitor di-N-acetylchitotriose. Particular attention is payed to the impact of the mapping, that is, the selection of AT and coarse-grained residues, on the binding free energy. It is shown that, in spite of small variations of the binding free energy with respect to the active site resolution, the separate contributions coming from different energetic terms (such as electrostatic and van der Waals interactions) manifest a stronger dependence on the mapping, thus pointing to the existence of an optimal level of intermediate resolution.
Topics: Animals; Avian Proteins; Binding Sites; Chickens; Female; Glycoside Hydrolase Inhibitors; Ligands; Models, Molecular; Muramidase; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Static Electricity; Substrate Specificity; Thermodynamics; Trisaccharides
PubMed: 32525263
DOI: 10.1002/prot.25954 -
The Biochemical Journal Mar 1982A glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) was isolated from calf rennet. This lysozyme was also present in abomasal secretions...
A glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) was isolated from calf rennet. This lysozyme was also present in abomasal secretions from calf and adult cattle. Multiple molecular forms revealed by electrofocusing might be artefacts. The main enzyme form had Mr approx. 15 000, pH optimum 5.0, pI7.5, and a remarkable conformation stability. Competitive inhibition was observed with both N-acetylglucosamine and N-acetylmuramic acid, with apparent Ki values of 29 mM and 2.4 mM respectively. The isolated enzyme also displayed significant chitinase activity.
Topics: Animals; Cattle; Chymosin; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Muramidase; Protein Conformation
PubMed: 6807289
DOI: 10.1042/bj2010661 -
Nature Communications Mar 2020We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states....
We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.
Topics: Bacteriophage T4; Biocatalysis; Crystallography, X-Ray; Fluorescence Resonance Energy Transfer; Molecular Dynamics Simulation; Monte Carlo Method; Muramidase; Mutagenesis, Site-Directed; Protein Structure, Tertiary; Viral Proteins
PubMed: 32144241
DOI: 10.1038/s41467-020-14886-w