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Prof. Jörg Standfuss

Paul Scherrer Institute, Villigen 

Dynamics of bacteriorhodopsin activation studied at synchrotrons and X-ray lasers 

X-ray free electron lasers offer exciting new opportunities to study the structural dynamics of proteins by time-resolved serial crystallography. By integrating sample efficient high viscosity injectors into pump probe setups, it is now possible to determine whole series of structural snapshots and assemble them to molecular movies of proteins in action. 

Based on our recent studies of the light-driven proton pump bacteriorhodopsin (bR), I will outline the possibilities but also the challenges that have to be overcome before we can routinely study atomic rearrangements in proteins at ambient temperature and in real time. One of the current bottlenecks is that access to X-ray lasers will remain scarce for the foreseeable future. To allow experiments at synchrotron sources, we have adapted high viscosity injector systems to carry out routine room-temperature serial millisecond crystallography at the Swiss Light Source (SLS) (1). Pilot experiments at the Swiss Free Electron Laser (SwissFEL) highlight the importance of careful sample preparation to make the most of the new femtosecond X-ray laser sources. 

Molecular movies of structural changes ranging from the femtosecond to the millisecond regime allowed us to study the proton transfer steps in bR with astounding detail. Mechanistically bR can be divided into an extracellular half and a cytoplasmic half with the retinal chromophore positioned roughly in the middle of the cellular membrane. The first principal step in the pumping mechanism is the light induced isomerization of retinal in the femtosecond range, which provides the energy for the reaction (2). In the second step, the energy is used to change the protein conformation within microseconds to allow proton release from the retinal Schiff base towards the extracellular release group via a water mediated hydrogen-bonding network (3). In the third principal step, the protein changes again after several milliseconds to allow uptake of a proton from the intracellular side of the membrane (4). These sequential rearrangements throughout the bR photocycle follow the basic predictions of an alternate access model and provide a template to understand the principal transport steps in other membrane pumps. 

References 

1. Weinert T, et al. (2017) Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons. Nature Communications 8(1):542. 

2. Nogly P, et al. (2018) Retinal isomerization in bacteriorhodopsin captured by a femtosecond X-ray laser. Science aat0094:10.1126–science.aat0094. 

3. Nango E, et al. (2016) A three-dimensional movie of structural changes in bacteriorhodopsin. Science 354(6319):1552–1557. 

4. Weinert T, et al., Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography, under evaluation