Studying small and fast
Studying molecular mechanisms behind the protein function is challenging due to the high spatiotemporal resolution needed for a detailed understanding of complex protein dynamics. The recent development of X-ray Free Electron Laser (XFEL) facilities opens new opportunities for dynamic structural biology. The intense and short 10-70 fs X-ray pulses enable pump-probe experiments with unprecedented temporal resolution. Time-resolved serial femtosecond crystallography (TR-SFX) can provide atomic-level details of proteins in action while probing events at femtosecond to millisecond timescales after activation.
Capturing protein in motion
Our group builds particularly on the recent experiments that focused on the dynamics of bacteriorhodopsin. This work demonstrated the sample-efficient TR-SFX at XFELs with high-viscosity injectors while providing unique insights into the activation and photocycle dynamics . Bacteriorhodopsin was also used to perform serial crystallography experiments at the synchrotron. Combined with a pump laser, it provides an excellent tool to probe slower protein dynamics at more widely available X-ray sources (TR-SSX) .
The combination of TR-SFX and TR-SSX enabled us to unravel the complex structural dynamics that drive chloride transport in halorhodopsin from Nonlabens marinus, NmHR . This comprehensive study was complemented by spectroscopy and QM/MM calculations to describe the mode of light energy utilization in transport initiation, the mechanism controlling the unidirectionality of the transport, and the tracing of the transport pathway.
Overview of major events during chloride transport by NmHR captured by time-resolved serial X-ray crystallography. For details, check our publication Mous et al. Science 2022.
In collaboration with the Gebhard Schertler and Valerie Panneels team from the Paul Scherrer Institute, we used the TR-SFX approach to gain insights into the first molecular events in vision .
Changes of chromophore binding in bovine rhodopsin probed by TR-SFX. Interactions between the retinal and the binding-pocket residues are substantially reduced by 1 ps after photoactivation in this G-protein coupled receptor. For details, check our publication Gruhl et al. Nature 2023.
Understanding signal conversion in receptors at the atomic level
The question at the heart of research at our Dioscuri Center is how different input signals (light, pH change, ligand binding, etc.) are converted at the molecular level to lead to different output signals. Recently, we started to study Light-Oxygen-Voltage (LOV) proteins. These act as blue light antennae and activate various effector proteins such as kinases, phosphatases, phosphodiesterases, and many more. Due to their universal action, LOV proteins found application as optogenetic tools to control the physiology of cells with light .
Photoactivation of the LOV domain leads to the formation of a covalent bond between Cys residue and the chromophore, followed by the activation of effector proteins with various functions. See our review Flores-Ibarra et al. JMB 2024.
Our first insights with the use of TR-SSX show the slow dynamics of the LOV domain and the structural changes associated with the initial stages of signal transduction .
TR-SSX experiment on LOV domain shows the structural dynamics associated with signal transduction in milliseconds after photoactivation. See our paper Gotthard et al. IUCrJ 2024 (a) (accepted)
We also used the protein as a tool to demonstrate the first successful TR-SFX experiments at XFEL with the use of fixed target sample delivery as a way to reduce sample requirements for this type of experiment drastically .
Different experimental setups were tested before we successfully demonstrated the TR-SFX experiment with LOV crystals delivered on a fixed target to drastically reduce sample consumption. See our paper Gotthard et al. IUCrJ 2024 (b)
Other exciting questions to explore include the ultrafast protein activation mechanism with the creation of a covalent thioether link to the chromophore, signal transduction to various effector domains, and the molecular mechanisms in the effector domains of different functions.
Studying dynamics of non-photoactive proteins
We extend TR-SFX experiments to non-photoactive proteins such as ionotropic receptors, using fast mixing techniques or photoswitching ligands to study structural dynamics at the atomic level, also in the case of medically relevant receptors.