Studying protein–ligand interactions using protein crystallography

Protein X-ray crystallography is now a relatively high-throughput technique that plays a major role in structure-based drug design programs where the method of soaking small organic ligands into protein crystals is used to confirm or identify binding modes. In this review, we discuss some experimental approaches for ‘soaking-in’ ligands into protein crystals where the major problem is poor solubility of the ligand. We also provide examples showing how crystal soaking can be used as a technique for establishing ligand binding strength: K c (the apparent ligand–protein dissociation constant in the crystal), which for some proteins at least, is found to be very similar to the solution K d. Kinetic effects are also found to be important and the rate at which ligands soak into crystals is shown to vary by orders of magnitude from 10−1 to 107 s depending on the system.