muramidase and succinimide

Amyloid fibrillation is closely associated with a series of neurodegenerative diseases. According to that, the intermediate soluble oligomers and protofibrils are more toxic; reducing their concentrations in protein solutions by accelerating fibrillation is believed as a feasible strategy for treatment or remission of the diseases. Using hen egg-white lysozyme (HEWL) as a model protein, the promotion effect of succinimide was revealed by a series of experiments, e.g., atomic force microscopy (AFM), thioflavin T (ThT) fluorescence assay, Far-UV circular dichroism (CD) and Raman spectroscopy, and modeling the effect of succinimide-like derivative intermediates of intramolecular deamidation of the backbone during amyloid fibrillation. The AFM measurement confirmed that succinimide effectively accelerated the morphological changes of HEWL, while at the molecular level, the accelerative transformation of protein secondary structures was also clarified by ThT fluorescence assay and Far-UV CD spectroscopy. The incubation time-dependent Raman spectroscopy further revealed that the direct transformation from α-helices to organized β-sheets occurred upon skipping the intermediate random coils under the action of succinimide. This "bridge" effect of succinimide was attributed to its special influence on disulfide bonds. In the presence of succinimide in protein solutions, the native disulfide bonds of lysozyme could be broken more efficiently and quickly within hydrolysis, resulting in exposure of the buried hydrophobic residues and accelerating the formation of cross β-sheet structures. The present investigation provides very useful information for understanding the effect of intramolecular deamidation on the whole amyloid fibrillation.

Topics: Amyloid; Animals; Chickens; Circular Dichroism; Microscopy, Atomic Force; Muramidase; Protein Conformation, beta-Strand; Succinimides

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.

Topics: Anti-Infective Agents; Calorimetry, Differential Scanning; Cell Wall; Circular Dichroism; Escherichia coli; Isoenzymes; Muramidase; Spectrometry, Fluorescence; Succinimides; Thermodynamics

Protein chemical degradations occur naturally into living cells as soon as proteins have been synthesized. Among these modifications, deamidation of asparagine or glutamine residues has been extensively studied, whereas the intermediate state, a succinimide derivative, was poorly investigated because of the difficulty of isolating those transient species. We used an indirect method, a limited thermal treatment in the dry state at acidic pH, to produce stable cyclic imide residues in hen lysozyme molecules, enabling us to examine the structural and functional properties of so modified proteins. Five cyclic imide rings have been located at sites directly accessible to solvent and did not lead to any changes in secondary or tertiary structures. However, they altered the catalytic properties of lysozyme and significantly decreased the intrinsic stability of the molecules. Moreover, dimerization occurred during the treatment, and this phenomenon was proportional to the extent of chemical degradation. We propose that succinimide formation could be responsible for covalent bond formation under specific physicochemical conditions that could be found in vivo.

Topics: Animals; Catalysis; Chickens; Hot Temperature; Hydrogen-Ion Concentration; Muramidase; Protein Multimerization; Protein Structure, Tertiary; Succinimides; Swine

The fragmentation behavior of a novel thiourea-based cross-linker molecule specifically designed for collision-induced dissociation (CID) MS/MS experiments is described. The development of this cross-linker is part of our ongoing efforts to synthesize novel reagents, which create either characteristic fragment ions or indicative constant neutral losses (CNLs) during tandem mass spectrometry allowing a selective and sensitive analysis of cross-linked products. The new derivatizing reagent for chemical cross-linking solely contains a thiourea moiety that is flanked by two amine-reactive N-hydroxy succinimide (NHS) ester moieties for reaction with lysines or free N-termini in proteins. The new reagent offers simple synthetic access and easy structural variation of either length or functionalities at both ends. The thiourea moiety exhibits specifically tailored CID fragmentation capabilities--a characteristic CNL of 85 u--ensuring a reliable detection of derivatized peptides by both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry and as such possesses a versatile applicability for chemical cross-linking studies. A detailed examination of the CID behavior of the presented thiourea-based reagent reveals that slight structural variations of the reagent will be necessary to ensure its comprehensive and efficient application for chemical cross-linking of proteins.

Topics: Animals; Chickens; Cross-Linking Reagents; Gonadotropin-Releasing Hormone; Muramidase; Peptide Fragments; Protein Conformation; Proteins; Substance P; Succinimides; Tandem Mass Spectrometry; Thiourea

The isomerization of Asp101 to isoaspartate autocatalytically proceeds via a succinimide intermediate in hen egg-white lysozyme at a mildly acidic condition. The crystal structures of succinimide and isoaspartate forms of the lysozyme proteins, each complexed with a tri-N-acetylchitotriose ligand, have been determined at 1.8 A resolution, and distinctively elucidate coplanar cyclic aminosuccinyl and beta-linked isoaspartyl residues. Compared with the liganded native protein with normal Asp101, succinimide 101 protrudes toward the ligand, and isoaspartate 101 extends away from the ligand. The formations of these residues caused the loss of three hydrogen-bonds between the ligand and the side-chains of Asp101 and Asn103 along with 0.5 A displacement of the ligand location.

Topics: Animals; Aspartic Acid; Chickens; Crystallography, X-Ray; Egg White; Isoenzymes; Models, Molecular; Muramidase; Succinimides; Trisaccharides

Topics: Chymotrypsin; Circular Dichroism; Detergents; Indicators and Reagents; Muramidase; Protein Binding; Proteins; Sodium Dodecyl Sulfate; Sodium Iodide; Spectrometry, Fluorescence; Subtilisins; Succinimides