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Structure-based screening of binding affinities via small-angle X-ray scattering

Title data

Chen, Po-Chia ; Masiewicz, Pawel ; Perez, Kathryn ; Hennig, Janosch:
Structure-based screening of binding affinities via small-angle X-ray scattering.
In: IUCrJ. Vol. 7 (2020) Issue 4 . - pp. 644-655.
ISSN 2052-2525
DOI: https://doi.org/10.1107/S2052252520004169

Abstract in another language

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 102 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.

Further data

Item Type: Article in a journal
Refereed: Yes
Keywords: drug discovery; molecular recognition; solution scattering; structural biology; SAXS
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry with an Emphasis on Biophysical Chemistry > Chair Biochemistry with an Emphasis on Biophysical Chemistry - Univ.-Prof. Dr. Janosch Hennig
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 06 Oct 2021 09:44
Last Modified: 06 Oct 2021 10:50
URI: https://eref.uni-bayreuth.de/id/eprint/67203