Change search
ReferencesLink to record
Permanent link

Direct link
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
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
Show others and affiliations
2013 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 120, 18-23 p.Article in journal (Refereed) Published
Abstract [en]

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.
Keyword [en]
Alzheimer's disease, Affibody molecule, Copper ion, Binding constant, Protein engineering, Peptide aggregation
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-119725DOI: 10.1016/j.jinorgbio.2012.11.005ISI: 000315252300003ScopusID: 2-s2.0-84871389016OAI: diva2:612789
Swedish Research CouncilVinnova

QC 20130325

Available from: 2013-03-25 Created: 2013-03-21 Last updated: 2014-02-07Bibliographically approved
In thesis
1. Chemical Engineering of Small Affinity Proteins
Open this publication in new window or tab >>Chemical Engineering of Small Affinity Proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS).

In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I).

In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III).

In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V).

Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. viii, 79 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:3
Affibody molecules, albumin binding domain, ligation, protein synthesis, solid phase peptide synthesis
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-141014 (URN)978-91-7595-004-4 (ISBN)
Public defence
2014-03-07, FR4 (Oskar Klein), AlbaNova Universitetscentrum, Stockholm, 10:00 (English)

QC 20140207

Available from: 2014-02-07 Created: 2014-02-05 Last updated: 2014-02-07Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Search in DiVA

By author/editor
Lindgren, JoelSegerfeldt, PatrikEriksson Karlström, Amelie
By organisation
Molecular Biotechnology (closed 20130101)
In the same journal
Journal of Inorganic Biochemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 54 hits
ReferencesLink to record
Permanent link

Direct link