Solution-phase, time-resolved structure of proteins from single-particle X-ray diffraction

The dynamics, conformational heterogeneity, and cellular conditioning of biomolecules is often crucial for their function.

However, methods to resolve these quantities in near-native solution conditions and with structural resolution are currently not available.We propose to pioneer structure determination of single proteins in solution using X-ray diffraction at Free Electron Lasers.

Key to this is a new liquid jet with ultrashort (50 nm) pathlength and unprecedented shot-to-shot stability, which will enable us to detect the diffraction from individual large protein complexes (>1 MDa) in solution.

We propose to develop a full Bayesian theoretical framework to maximize the extraction of useful information from the data, and to utilize time-resolved X-ray scattering to resolve a complete enzymatic mechanism. We will first validate the method using DNA origami clusters.

Then we will use it to study photoprotection in cyanobacterial photosynthesis by pioneering structure detection of single phycobilisome antenna proteins (PBS), resolving the structural mechanism of photoexcitation in orange carotenoid proteins (OCP), and unveiling single-molecular, time-resolved structural changes in OCP-PBS complexes.

Together, this may break considerable new biological ground.The highly collaborative project may establish single-protein structure determination at the Eu-XFEL. This has high scientific significance and will open up for numerous studies in biology and medicine.