Impact of subunit linkages in an engineered homodimeric binding protein to α-synuclein

Gauhar, Aziz; Gremer, Lothar; Hoyer, Wolfgang; Mirecka, Ewa; Shaykhalishahi, Hamed

doi:10.1093/protein/gzu047

Journal Article

FZJ-2014-05666

Impact of subunit linkages in an engineered homodimeric binding protein to α-synuclein

Gauhar, A. ; Shaykhalishahi, H. ; Gremer, L.FZJ* ; Mirecka, E. ; Hoyer, W.FZJ*

2014
Oxford Univ. Press Oxford

Protein engineering design and selection 27(12), 473-479 (2014) [10.1093/protein/gzu047]

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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.

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