Engineered non-fluorescent Affibody molecules facilitate studies of the amyloid-beta (A beta) peptide in monomeric form: Low pH was found to reduce A beta/Cu(II) binding affinity
2013 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 120, 18-23 p.Article in journal (Refereed) Published
Aggregation of amyloid-beta (A beta) peptides into oligomers and amyloid plaques in the human brain is considered a causative factor in Alzheimer's disease (AD). As metal ions are over-represented in AD patient brains, and as distinct A beta aggregation pathways in presence of Cu(II) have been demonstrated, metal binding to A beta likely affects AD progression. A beta aggregation is moreover pH-dependent, and AD appears to involve inflammatory conditions leading to physiological acidosis. Although metal binding specificity to A beta varies at different pH's, metal binding affinity to A beta has so far not been quantitatively investigated at sub-neutral pH levels. This may be explained by the difficulties involved in studying monomeric peptide properties under aggregation-promoting conditions. We have recently devised a modified Affibody molecule, Z(A beta 3)(12-58), that binds A beta with sub-nanomolar affinity, thereby locking the peptide in monomeric form without affecting the N-terminal region where metal ions bind. Here, we introduce non-fluorescent A beta-binding Affibody variants that keep A beta monomeric while only slightly affecting the A beta peptide's metal binding properties. Using fluorescence spectroscopy, we demonstrate that Cu(II)/A beta(1-40) binding is almost two orders of magnitude weaker at pH 5.0 (apparent K-D = 51 mu M) than at pH 7.3 (apparent K-D = 0.86 mu M). This effect is arguably caused by protonation of the histidines involved in the metal ligandation. Our results indicate that engineered variants of Affibody molecules are useful for studying metal-binding and other properties of monomeric A beta under various physiological conditions, which will improve our understanding of the molecular mechanisms involved in AD.
Place, publisher, year, edition, pages
2013. Vol. 120, 18-23 p.
Alzheimer's disease, Affibody molecule, Copper ion, Binding constant, Protein engineering, Peptide aggregation
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:su:diva-88710DOI: 10.1016/j.jinorgbio.2012.11.005ISI: 000315252300003OAI: oai:DiVA.org:su-88710DiVA: diva2:612826
FunderSwedish Research CouncilVinnova