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.
Highlights
The function of a protein is determined by its structure which defines specific properties in a biological context and its dynamic interactions with diverse partners such as ions, lipids, hormones, other proteins, and nucleic acids
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-Ray Free Electron Lasers (X-FELs)
The emerging TR-SFX method for the study of structural dynamics is an unique opportunity for the membrane protein field
Summary
The function of a protein is determined by its structure which defines specific properties in a biological context and its dynamic interactions with diverse partners such as ions, lipids, hormones, other proteins, and nucleic acids. About 90% of all structures in the Protein Data Bank have been obtained by X-ray crystallography This method relies on crystals to amplify the structural information and reach sufficient signal to noise ratios. This method delivers the best results with large, well-ordered crystals that are often difficult to obtain, especially for membrane proteins. Radiation damage poses another fundamental barrier that limits the achievable data quality and may alter the structure of proteins in possibly misleading ways. A number of major breakthroughs have been achieved, including a 1.9 Aresolution structure of lysozyme as the first high-resolution structure from SFX (Boutet et al, 2012)
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