Crystallography at non-ambient conditions

Crystallography at non-ambient conditions

The functions of proteins are driven by their structure and the conformational space they can sample. The free-energy landscape of proteins is defined by multiple variables, such as protein concentration, ligands and solvents, but also thermodynamic parameters such as temperature and pressure. However, protein dynamics are most often only analyzed at ambient conditions (room temperature or physiological temperatures, e.g. 37C, and atmospheric pressure) and, thus, a large part of the free-energy landscape remains unstudied.

Pressure as physical variable is known to have a strong influence on the protein fold and especially on enzymatic activity. We are interested in studying the effects of pressure and temperature on protein stability and function at the atomic level with high-pressure X-ray crystallography. For this purpose, we have developed a new high-pressure cell that is pressurized with helium up to 120 MPa. With this device we are able to study several aspects that are important to understand the fundamental principles of the protein structure. We want to answer the question how proteins adapt to natural high-pressure habitats such as the deep sea, want to use pressure to better understand enzymatic mechanisms, but also want to explore the influence of pressure on industrially relevant enzymes. The latter will provide the fundamental basis for the rational design of a new generation of pressure-adapted enzymes, thereby fully exploring pressure as profitable process parameter for biotechnological applications.

Protein dynamics allow the protein to sample different parts of their energetic landscape and are the basis of protein function, whether it is binding to ligands or facilitating enzymatic reactions. Motions on the femtosecond to millisecond scale determine how proteins behave ranging from bond vibrations to large scale domain movements. X-ray crystallography offers the highest spatial resolution of macromolecules. However, these structures represent only a temporally averaged snapshot of the most populated conformations. Various other methods are available that probe protein dynamics experimentally. But they lack high spatial resolution. Time-resolved X-ray crystallography has begun to discern transient conformations of macromolecular structures. Here, the system is removed from its equilibrium by laser light or by addition of ligands. While the temporal resolution of ligand binding is generally hampered by the diffusion of ligands into the crystal, mostly to the millisecond range, activation by laser light can resolve femtosecond processes. Infrared laser-induced temperature jump combined with X-ray solution scattering demonstrated to be able to unveil hidden conformational dynamics in an enzyme. In contrast to solution scattering, X-ray crystallography provides a much higher spatial resolution down to the atomic level. Therefore, we combine temperature jump of protein crystals with pink beam serial crystallography to remove proteins from their conformational equilibrium. We take snapshots of the structure from nano- to milliseconds to follow the changes in the protein crystals as it returns from an energetically excited to its ground state at ambient temperatures with the aim to uncover dynamically linked networks of amino acids that could be important for protein function. This method has the potential to shed light on the protein dynamics of virtually any crystallizable protein at high spatial and temporal resolution.