A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers.

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

Increasing the chemical space of proteins in living cells via genetic code expansion.

In recent years it has become possible to genetically encode an expanded set of designer amino acids with tailored chemical and physical properties (dubbed unnatural amino acids, UAAs) into proteins in living cells by expanding the genetic code. Together with developments in chemistries that are amenable to and selective within physiological settings, these strategies have started to have a big impact on biological studies, as they enable exciting in cellulo applications. Here we highlight recent advances to covalently stabilize transient protein-protein interactions and capture enzyme substrate-complexes in living cells using proximity-triggered and residue-selective photo-induced crosslinking approaches. Furthermore, we describe recent efforts in controlling enzyme activity with photocaged UAAs and in extending their application to a variety of enzymatic scaffolds. In addition, we discuss the site-specific incorporation of UAAs mimicking post-translational modifications (PTMs) and approaches to generate natively-linked ubiquitin-protein conjugates to probe the role of PTMs in modulating complex cellular networks.