muramidase and indole

Amyloid aggregation of polypeptides is related to a growing number of pathologic states known as amyloid disorders. There is a great deal of interest in developing small molecule inhibitors of the amyloidogenic processes. In the present article, the inhibitory effects of some indole derivatives on amyloid fibrillation of hen egg white lysozyme (HEWL) are reported. Acidic pH and high temperatures were used to drive HEWL towards amyloid formation. A variety of techniques, ranging from thioflavin T fluorescence and Congo red absorbance assays to far-UV CD and transmission electron microscopy, were employed to characterize the HEWL fibrillation process. Among the indole derivatives tested, indole 3-acetic acid, indole 3-carbinol and tryptophol had the most inhibitory effects on amyloid formation, indole and indole 3-propionic acid gave some inhibition, and indole aldehyde and tryptophan showed no significant inhibition. Although indoles did not protect the HEWL native state from conformational changes, they were effective in diminishing HEWL amyloid fibril formation, delaying both the nucleation and elongation phases. Disaggregation of previously formed HEWL amyloid fibrils was also enhanced by indole 3-acetic acid. Various medium conditions, such as the presence of different anions and alcoholic cosolvents, were explored to gain an insight into possible mechanisms. These observations, taken together, suggest that the indole ring is likely to play the main role in inhibition and that the side chain hydroxyl group may contribute positively, in contrast to the side chain carbonyl and intervening methylene groups.

Topics: Amyloid; Animals; Binding Sites; Chickens; Circular Dichroism; Hydrogen-Ion Concentration; Indoleacetic Acids; Indoles; Kinetics; Microscopy, Electron, Transmission; Models, Molecular; Molecular Structure; Muramidase; Protein Binding; Protein Conformation; Temperature

A new protocol is described for the isotope (15N and 13C,15N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. 1H,13C and 15N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the Cgamma resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.

Topics: Animals; Carbon Isotopes; Chickens; Escherichia coli; Hydrophobic and Hydrophilic Interactions; Indoles; Methylation; Muramidase; Mutation; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Recombinant Proteins; Tryptophan

Two novel luminescent rhenium(I) diimine indole complexes have been designed and their properties studied; these conjugates can be recognised by indole-binding proteins including bovine serum albumin, lysozyme and tryptophanase.

Topics: Animals; Cattle; Electrochemistry; Imines; Indoles; Ligands; Luminescence; Models, Molecular; Molecular Structure; Muramidase; Organometallic Compounds; Photochemistry; Protein Binding; Rhenium; Serum Albumin, Bovine; Tryptophanase

We have investigated the magnitude and timescale of fluctuations within the core of a protein using the exchange kinetics of indole and benzene binding to engineered hydrophobic cavities in T4 lysozyme. The crystal structures of variant-benzene complexes suggest that relatively large scale fluctuations (1-2 angstrom) of backbone atoms are required for entry of these ligands into the core. Nonetheless, these ligands enter the cavities rapidly, with bimolecular rate constants of approximately 10(6)-10(7) M(-1) s(-1) and a low activation barrier, 2-5 kcal mol(-1). These results suggest that protein cores undergo substantial fluctuations on the millisecond to microsecond timescale and that entry of small molecules into protein interiors is not strongly limited by steric occlusion.

Topics: Benzene; Binding Sites; Energy Transfer; Indoles; Magnetic Resonance Spectroscopy; Models, Molecular; Muramidase; Protein Conformation; Proteins; Recombinant Proteins

To better understand the role of shape complementarity in ligand binding and protein core interactions, the structures have been determined of a set of ligands bound within a cavity-containing mutant of T4 lysozyme. The interior cavity is seen to consist of two parts that respond very differently to the binding of ligands. First, there is a relatively rigid region that does not relax significantly upon binding any ligand. Second, there is a more flexible region that moves to various extents in response to binding the different ligands. The part of the binding site that remains rigid is characterized by low temperature factors and strong protection from hydrogen exchange. This part of the site appears to be primarily responsible for discriminating between ligands of different shape (i.e., for determining specificity). The more flexible region, characterized by relatively high temperature factors and weak protection from hydrogen exchange, allows some promiscuity in binding by undergoing variable amounts of deformation at essentially the same energetic cost. This linkage between the dynamic information represented by crystallographic temperature factors and hydrogen-exchange behavior on the one hand, and structural plasticity in response to ligand binding on the other hand, suggests a way to improve our understanding of steric interactions in protein cores and protein-ligand binding sites. Ligand design and packing algorithms might take advantage of this information, requiring complementary interactions where the protein is rigid and allowing some overlap in regions where the protein is flexible.

Topics: Bacteriophage T4; Benzene Derivatives; Benzofurans; Computer Graphics; Crystallography, X-Ray; Hydrogen Bonding; Indenes; Indoles; Ligands; Models, Molecular; Muramidase; Protein Binding; Protein Conformation; Xylenes