High-pressure macromolecular crystallography as a tool for studying protein flexibility.

Protein function depends on their conformational flexibility. Complementary to temperature-dependent studies, high-pressure (HP) macromolecular crystallography is well suited to exploring the conformational space of proteins at atomic resolution. We have developed a new HP cell concept for macromolecular X-ray crystallography that overcomes the limitations of current devices. It consists of a single-crystalline sapphire capillary, which is loaded with multiple crystals and pressurized with helium at freely adjustable gas pressures of up to 120 MPa. Studying five different proteins, we generally observed a compression of the protein structures and a decrease in thermal displacement parameters at high pressure. Structurally, observations included the compression of a central tunnel of a biotechnologically relevant enzyme and the stabilization of ligand binding in the membrane protein photosynthetic reaction center of Cereibacter sphaeroides. For two pharmaceutically relevant proteins, the human kinase DRAK2 and SARS-CoV-2 main protease, we identified concerted movements of subdomains. Such knowledge is essential for predictions of protein dynamics and defined conformational states, offering the potential for improved structure-based drug development.