We investigated the structural effects induced by Al3+ on different beta-amyloid (Abeta) fragments at pH 7.4 and T=25 degrees C, with particular attention given to the sequences 1-40 and 1-42. Al3+ caused peptide enrichment in beta sheet structure and formation of solvent-exposed hydrophobic clusters. These intermediates evolved to polymeric aggregates which organized in fibrillar forms in the case of the Al3+-Abeta(1-42) complex. Comparative studies showed that Zn2+ and Cu2+ were much less efficient than Al3+ in stimulating the spontaneous aggregation/fibrillogenesis of Abetas. Studies with liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of Al3+-Abeta complexes, suggesting a major role of Al3+ in Abeta-induced cell dysfunction. Al3+ effects were abolished by desferrioxamine mesylate (DFO) only in solution. We concluded that, in vivo, DFO may act as a protective agent by preventing or reverting Abeta aggregation in the extracellular spaces.
Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of beta-sheets into alpha-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.
SynopsisThe conformational properties of synthetic S-peptide analogs, in which the residues in the N-terminal sequence 1-6 were progressively deleted or replaced with amino acids of lower helical propensity, were studied by CD. Increasing the concentration of guanidine hydrochloride and decreasing the temperature were found to produce progressive destruction of ordered conformations, in parallel with the increasing solubility of the peptide unit, while increasing the concentration of trifluoroethanol and decreasing the temperature produced the opposite effect. The maximum helicity determined in these sets of experiments is found equal to or greater than that determined in the formation of the ribonuclease S' complexes.With some peptides the maximum value of predicted helical conformation is reached, and the tendency of tertiary structure to reduce the maximum possible helicity is evident. We discuss the validity of the procedure by which conformational information, drawn from measurements in helicogenic solvents, is related to the state in native protein.
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