We used multiple in vitro binding experiments to test binding site and affinity for two top hits from iDTP, namely the peroxisome proliferator-activated receptor gamma (PPARγ) and B-cell lymphoma 2 (Bcl-2).
Second, color-coding of proteins with QDs of different emission wave lengths offers the unique opportunity to monitor multiple binding events in a single experiment on a single bead.
Kinetic experiments show the multiple binding modes of HD1 and NU172, which both interact with multiple sites of thrombin with low nanomolar affinities and show little specificity of interaction for prothrombin vs. thrombin.
It is therefore reasonable to think of even more complicated experiments, aiming to discover multiple binding specificities of protein complexes or even whole mixtures of proteins at once.
Most importantly, the authors measure a ten times higher affinity between the complexin accessory helix and blocked SNARE complex by ITC, and another ITC experiment provides evidence for multiple binding sites of complexin to SNARE complex.
Recent docking experiments with α-lobeline have predicted multiple binding sites, consistent with a non-specific chemical chaperone.
The high concentration of HI-6 in the soaking experiment could in theory lead to multiple binding modes (i.e., HI-6 occupies low- and high-affinity sites).
It is obvious from this analysis that a high proportion of previous SH3 binding experiments assessed ligands with potential to have multiple ligand binding modes.
In 2011, multiple experiments by the Rothman, Reinisch, and Tareste groups published: ITC binding experiments, crystal structures with a mutant of complexin in complex with a truncated SNARE complex, solution (bulk) FRET data, and surface force measurements.
Analysis of inferred active-site residues (D7, E60, K102, D132, and D135) by mutagenesis and metal rescue experiments specified residues that contribute to binding metal ions and that multiple binding sites are probably involved.
