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@ARTICLE{Gauhar:172156,
author = {Gauhar, Aziz and Shaykhalishahi, Hamed and Gremer, Lothar
and Mirecka, Ewa and Hoyer, Wolfgang},
title = {{I}mpact of subunit linkages in an engineered homodimeric
binding protein to α-synuclein},
journal = {Protein engineering design and selection},
volume = {27},
number = {12},
issn = {0269-2139},
address = {Oxford},
publisher = {Oxford Univ. Press},
reportid = {FZJ-2014-05666},
pages = {473-479},
year = {2014},
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.},
cin = {ICS-6},
ddc = {540},
cid = {I:(DE-Juel1)ICS-6-20110106},
pnm = {453 - Physics of the Cell (POF2-453)},
pid = {G:(DE-HGF)POF2-453},
typ = {PUB:(DE-HGF)16},
UT = {WOS:000345837300002},
doi = {10.1093/protein/gzu047},
url = {https://juser.fz-juelich.de/record/172156},
}