Time-resolved structural studies with serial crystallography: A new light on retinal proteins

Abstract

Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.

The light‐driven sodium ion pump: A new player in rhodopsin research

“…Although a number of recent studies have given us a hint about the dynamic properties of KR2 [24,33,54], we know almost nothing about (1) the detailed structural changes in the Schiff base region À including the formation of transient Na þ binding site in the O-intermediate À and (2) the large conformational change of the protein backbone during the photocycle. Similar to the cases of BR and HR [55][56][57][58], structural and spectroscopic techniques such as time-resolved (TR) macromolecular crystallography (e.g. Laue method and TR-serial femtosecond crystallography) and FTIR spectroscopy are suitable to study detailed structural changes inside the protein.…”

Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics

“…601 The static roomtemperature structures of an antagonist-bound angiotensin receptor 602 and of rhodopsin bound to visual arrestin 114 were solved by serial femtosecond X-ray crystallography.…”

Relativistic Ultrafast Electron Microscopy: Single-Shot Diffraction Imaging with Femtosecond Electron Pulses

“…Recently, single-shot diffraction imaging with ultrashort Xray pulses generated from free-electron lasers has facilitated the study of structural dynamics of irreversible processes in material samples [2] and the acquisition of direct structural information in chemistry and biology before sample damage [3]. However, ultrafast electron diffraction and microscopy (UED and UEM) with electron pulses are also very promising techniques for the study of ultrafast structural dynamics in materials because electrons are complementary to X-rays in a number of ways [4]:…”

“…Measurement using electrons is used to observe structural information of small or thin crystals, light-element materials, and gas phase samples [5]. (2) Electron imaging technology with high spatial resolution is well developed. (3) Femtosecond electron pulses are achievable using photoemission guns.…”