TY  - JOUR
AU  - Gauhar, Aziz
AU  - Shaykhalishahi, Hamed
AU  - Gremer, Lothar
AU  - Mirecka, Ewa
AU  - Hoyer, Wolfgang
TI  - Impact of subunit linkages in an engineered homodimeric binding protein to α-synuclein
JO  - Protein engineering design and selection
VL  - 27
IS  - 12
SN  - 0269-2139
CY  - Oxford
PB  - Oxford Univ. Press
M1  - FZJ-2014-05666
SP  - 473-479
PY  - 2014
AB  - 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.
LB  - PUB:(DE-HGF)16
UR  - <Go to ISI:>//WOS:000345837300002
DO  - DOI:10.1093/protein/gzu047
UR  - https://juser.fz-juelich.de/record/172156
ER  -