Fibrillation of Carrier Protein Albebetin and Its Biologically Active Constructs. Multiple Oligomeric Intermediates and Pathways
2004 (English)In: Biochemistry, ISSN 0006-2960, Biochemistry, Vol. 43, no 30, 9610-9619 p.Article in journal (Refereed) Published
We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-2 octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and -sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.
Place, publisher, year, edition, pages
2004. Vol. 43, no 30, 9610-9619 p.
IdentifiersURN: urn:nbn:se:umu:diva-14787DOI: 10.1021/bi0494121PubMedID: 15274615OAI: oai:DiVA.org:umu-14787DiVA: diva2:154459