Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser

Abstract

Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

“…Time-resolved crystallographic studies provided much insight into how proton pumping is achieved in bR. In particular they focused on the early events in the pumping cycle including the ultrafast process of energy capture through photoisomerization of retinal (Nogly et al 2018) and the proton release step from the retinal Schiff base (SB) towards the extracellular side of the membrane ). The fundamental piece missing in our dynamic view of proton transport is how the protein rearranges to reprotonate the SB from the cytoplasmic side to reload substrate for the next pumping cycle.…”

“…The purification and crystallization of bacteriorhodopsin were performed as described previously (Nogly et al 2018). Purple membranes from Halobacterium salinarum were solubilized overnight in the presence of 1.2% b-octylglucoside (Anatrace) and 50 mM sodium phosphate buffer pH 6.9 (GERBU).…”

“…The SFX dark-state model (PDB code 6G7H (Nogly et al 2018)) was directly refined against the light off data (Steady-SMX dark, Supplementary Table 1). Extrapolated data were calculated as described elsewhere (Nogly et al 2018). The activation level was determined by calculating extrapolated maps using the steady-SMX light data and the steady-SMX dark data at different activation levels in steps of 2 % until features of the dark-state appeared at the retinal, Arg 82 and in helices E, F and G. Based on this analysis an activation level of 38 % was chosen.…”

Ultracompact 3D microfluidics for time-resolved structural biology

“…Serial femtosecond crystallography (SFX) at X-ray free-electron lasers seeks to extend the sensitivity and applicability of X-ray diffraction to structural biology. It has allowed the observation of radiation-sensitive structures at the atomic scale [5][6][7] . The method greatly increases the exposure of a crystal beyond conventional limits, and without the need for cryogenic cooling, by using femtosecond-duration X-ray pulses that terminate before the onset of radiation damage 8 .…”

“…The short X-ray exposure times achieved in serial crystallography can be used to reveal time-resolved conformational dynamics of biological macromolecules and their role in catalysis, long-range structural interaction pathways, induced fit and selectivity in ligand binding, and allosteric control of conformational entropy 26 . To date, most time-resolved experiments use a pumpprobe scheme, in which an optical excitation pulse triggers the system at a prescribed time before the arrival of the X-ray measurement pulse 6,7,27 . However, only a small fraction of biological reactions are naturally induced by light, while most are initiated upon substrate binding.…”