Ligand binding and molecular recognition mechanisms often involve physical changes in the conformation or structural arrangement of participating macromolecules. Here, solution small-angle X-ray scattering (SAXS) provides a means to quantify structural change, as it yields the distances between all solute atoms as a 1D distribution. Additionally, mixtures of different populations can be decomposed by precise measurements across different relative concentrations. In the context of screening techniques, this in principle enables computations of effective binding affinities, as well as identification of antagonists together with complementary screening data. However, a lack of reference benchmarks hinders feasibility studies into the incorporation of SAXS within high throughput screening workflows for structural biology and drug discovery.
This study presents the use of 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 protein concentration and total exposure times exceed 20 seconds, the presented titration protocol at 0.5 mg/ml yield affinities comparable to isothermal titration calorimetry measurements. Estimated throughput ranges between 20~100 ligand titration per day at current synchrotron beamlines, with the limiting step imposed by sample handling and cleaning protocols.