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N-terminal engineering of amyloid-β-binding Affibody molecules yields improved chemical synthesis and higher binding affinity
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2010 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, no 12, 2319-2329 p.Article in journal (Refereed) Published
Abstract [en]

The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)

Place, publisher, year, edition, pages
2010. Vol. 19, no 12, 2319-2329 p.
Keyword [en]
amyloid, protein engineering, Alzheimer's disease, solid phase peptide synthesis, NMR spectroscopy
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-51261DOI: 10.1002/pro.511ISI: 000284793800006OAI: oai:DiVA.org:su-51261DiVA: diva2:385091
Note
authorCount :9Available from: 2011-01-11 Created: 2011-01-10 Last updated: 2017-12-11Bibliographically approved
In thesis
1. NMR studies on interactions between the amyloid β peptide and selected molecules
Open this publication in new window or tab >>NMR studies on interactions between the amyloid β peptide and selected molecules
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease is an incurable neurodegenerative disorder linked to the amyloid β (Aβ) peptide, a 38-43 residue peptide. The detailed molecular disease mechanism(s) is (are) unknown, but oligomeric Aβ structures are proposed to be involved.

In common for the papers in this thesis is interactions; interactions between Aβ(1-40) and selected molecules and metal ions. The purpose has been to find out more about the structural states that Aβ can adopt, in particular the β-sheet state, which probably is linked to the oligomeric structures. The methods used have been nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopy using Thioflavin T (ThT).

Upon addition of SDS/LiDS detergent or Congo red (CR) to Aβ(1-40), the initial random coil/PII-helix state was transformed into β-sheet and, in the case of detergent, a final α-helical state. In contrast to SDS/LiDS and CR, the dimeric Affibody molecule locks monomeric Aβ(1-40) in a β-hairpin state. It was found that by truncating the flexible N-terminal end of the Affibody molecule its affinity to Aβ was improved. The aggregation of Aβ(1-40) was further studied in the presence of a β-cyclodextrin dimer by a kinetic assay using ThT. Although having a weak dissociation constant in the millimolar range, the β-cyclodextrin dimer modified the aggregation pathways of Aβ.

Finally Aβ(1-40) was studied in presence of Cu2+ and Zn2+ at physiological and low pH. Cu2+ was observed to maintain its specific binding to Aβ when decreasing the pH to 5.5 while Zn2+ behaved differently. This could be of importance in the Alzheimer’s disease brain in which the environment can become acidic due to inflammation.       

In conclusion the results show that Aβ(1-40) is very sensitive to its environment, responding by adopting different conformations and aggregating in aqueous solutions. The β-sheet state is induced by varying molecules with different properties, properties that govern the final Aβ state.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 59 p.
Keyword
Amyloid β peptide, Alzheimer's disease, Aggregation, Oligomer, Amyloid, Interaction, Nuclear Magnetic Resonance Spectroscopy, Circular Dichroism Spectroscopy, Thioflavin T, Detergent, Congo red, Affibody, Cyclodextrin dimer, Metal ion
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-60346 (URN)978-91-7447-325-4 (ISBN)
Public defence
2011-10-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2011-09-15 Created: 2011-08-15 Last updated: 2011-09-02Bibliographically approved

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