Investigating the role of Greek key motifs of amylase in amyloid fibril formation

Introduction: Amyloid fibrils are a class of protein nanofibres that form via self-associating of protein into dimer and oligomers, which eventually produces condensed fibril forms. A process of protein fibrillation that causes intracellular or extracellular accumulation of insoluble protein deposits causes many important neurodegenerative diseases such as Alzheimer's, Huntington's chorea or Parkinson's. In most amyloid fibrils a conformational switching occurs from α-helix or random coil, to a β-sheet structure. To examine whether or not β-sheet containing proteins, are predisposed to form amyloid fibrils, α-amylase (containing three domaim A, B and C) from Bacillus sp. KR8104 was chosen as a model and its aggregation was studied in the presence and absence of C domain consisting of beta sheets in the form of Greek key (consists of four adjacent antiparallel strands) arrangements. Materials and methods: PCR was carried out to entirely remove the C domain of α-amylase. Based on the crystallography data and sequence analysis, specific primers were designed to delete the domain. The gene encoding a full length α-amylase (previously cloned into pET28a) was used as a templet in the PCR mixture. The PCR product obtained for truncated gene was digested and cloned into NcoI and XhoI restriction sites of an expression vector, pET28a. The constructed recombinant plasmid was transformed into E. coli BL21 and expressed under optimal condition (0.5 mM IPTG at 20 °C for 2 hours). Amyloid fibril formation of both constructs (with and without domain C) were optimized and then analyzed using thioflavin-T and red congo. Results: The native enzyme expressed as both soluble and inclusion bodies, but the construct with no c domain only expressed as inclusion bodies. Inclusion bodies from both constructs were examined for their ability to form amyloid fibrils using congo red. The inclusion bodies were then solubilized in Urea (8M), purified and refolded on an affinity column. The aggregation of both soluble proteins into amyloid fibrils were studied in 50 mM tris (pH 7) at 50 °C using thioflavin-T fluorescence assay. Results revealed differences between the complete and truncated protein. Conclusion: Our data suggest that the highly C terminal region (Greek key motif) of α-amylase may act as an intramolecular chaperone by protecting the hydrophobic domain from aggregation. Understanding the function of such chaperone-like parts of fibril-forming proteins may provide novel insights into the mechanism of amyloid formation.© 2016 Pu

کلیدواژه ها:

α-Amylase ، Domain C ، Amyloid fibrils