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@ARTICLE{Chen:448363,
author = {Chen, Po-chia and Masiewicz, Pawel and Perez, Kathryn and
Hennig, Janosch},
title = {{S}tructure-based screening of binding affinities via
small-angle {X}-ray scattering},
journal = {IUCrJ},
volume = {7},
number = {4},
issn = {2052-2525},
address = {Chester},
reportid = {PUBDB-2020-03391},
pages = {644 - 655},
year = {2020},
abstract = {Protein–protein and protein–ligand interactions often
involve conformational changes or structural rearrangements
that can be quantified by solution small-angle X-ray
scattering (SAXS). These scattering intensity measurements
reveal structural details of the bound complex, the number
of species involved and, additionally, the strength of
interactions if carried out as a titration. Although a core
part of structural biology workflows, SAXS-based titrations
are not commonly used in drug discovery contexts. This is
because prior knowledge of expected sample requirements,
throughput and prediction accuracy is needed to develop
reliable ligand screens. This study presents the use of the
histidine-binding protein (26 kDa) and other periplasmic
binding proteins to benchmark ligand screen performance.
Sample concentrations and exposure times were varied across
multiple screening trials at four beamlines to investigate
the accuracy and precision of affinity prediction. The
volatility ratio between titrated scattering curves and a
common apo reference is found to most reliably capture the
extent of structural and population changes. This obviates
the need to explicitly model scattering intensities of bound
complexes, which can be strongly ligand-dependent. Where the
dissociation constant is within 10$^2$ of the protein
concentration and the total exposure times exceed 20 s,
the titration protocol presented at 0.5 mg ml$^{−1}$
yields affinities comparable to isothermal titration
calorimetry measurements. Estimated throughput ranges
between 20 and 100 ligand titrations per day at current
synchrotron beamlines, with the limiting step imposed by
sample handling and cleaning procedures.},
cin = {EMBL-User},
ddc = {530},
cid = {I:(DE-H253)EMBL-User-20120814},
pnm = {6G3 - PETRA III (POF3-622)},
pid = {G:(DE-HGF)POF3-6G3},
experiment = {EXP:(DE-H253)P-P12-20150101},
typ = {PUB:(DE-HGF)16},
pubmed = {pmid:32695411},
UT = {WOS:000548507200007},
doi = {10.1107/S2052252520004169},
url = {https://bib-pubdb1.desy.de/record/448363},
}