title: Impact of subunit linkages in an engineered homodimeric binding protein to α-synuclein.
abstract: Aggregation of the protein α-synuclein (α-syn) has been implicated in Parkinson's disease and other neurodegenerative disorders, collectively referred to as synucleinopathies. The β-wrapin AS69 is a small engineered binding protein to α-syn that stabilizes a β-hairpin conformation of monomeric α-syn and inhibits α-syn aggregation at substoichiometric concentrations. AS69 is a homodimer whose subunits are linked via a disulfide bridge between their single cysteine residues, Cys-28. Here we show that expression of a functional dimer as a single polypeptide chain is achievable by head-to-tail linkage of AS69 subunits. Choice of a suitable linker is essential for construction of head-to-tail dimers that exhibit undiminished α-syn affinity compared with the solely disulfide-linked dimer. We characterize AS69-GS3, a head-to-tail dimer with a glycine-serine-rich linker, under oxidized and reduced conditions in order to evaluate the impact of the Cys28-disulfide bond on structure, stability and α-syn binding. Formation of the disulfide bond causes compaction of AS69-GS3, increases its thermostability, and is a prerequisite for high-affinity binding to α-syn. Comparison of AS69-GS3 and AS69 demonstrates that head-to-tail linkage promotes α-syn binding by affording accelerated disulfide bond formation.
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Aziz
authors.initials: A
authors.lastname: Gauhar
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Hamed
authors.initials: H
authors.lastname: Shaykhalishahi
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, 52425 Jülich, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Gremer
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors.forename: Ewa A
authors.initials: EA
authors.lastname: Mirecka
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, 52425 Jülich, Germany wolfgang.hoyer@uni-duesseldorf.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hoyer
mesh_terms: D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D051844:alpha-Synuclein / Q000737:chemistry*
chemical_list: D004220:Disulfides; D010455:Peptides; D011994:Recombinant Proteins; D051844:alpha-Synuclein
keywords: alternative scaffold; amyloid; disulfide bond; α-synuclein; β-wrapin