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.

FIG. 1.

Scheme of pump–probe time-resolved SFX experimental setup for retinal protein dynamics study. The figure shows the overall setup containing the optical pump laser (rainbow), the probe X-ray FEL pulse (in blue), and the detectors. The high X-FEL intensities used in the “diffract before destroy” regime of SFX require a constant delivery of crystals at room temperature, precisely aligned with the path of the pump laser which is set at the wavelength specific for the retinal protein photoactivation. After a precise time-delay, the X-FEL pulse is delivered in the interaction region to record the structural information about the induced changes. To date, among the various sample delivery modes available for SFX, including fixed target wafers (a) with arrays of windows with painted 2D or 3D crystals in a glucose-containing solution, only the microjet (b) system delivering a liquid stream of submicron crystals is used for TR-SFX.

FIG. 2.

Time-resolved serial femtosecond crystallography (TR-SFX) studies along the photocycle of the retinal proteins rhodopsin and bacteriorhodopsin (bR). The advent of the X-FEL as a source of very intense X-ray pulses enables to perform now ultrafast time-resolved structural studies at the atomic level. The left panel shows the bacteriorhodopsin photocycle going through the K to O spectral intermediates (Lorenz-Fonfria and Kandori, 2009) and recycling back to the dark state in a few milliseconds. Without a X-FEL (orange box, TR-SFX), the first intermediates could theoretically not be structurally determined, and the later intermediates being in the time-range of synchrotron time-delays (blue box, TR-SMX (time-resolved serial millisecond crystallography)). The right panel shows the photoactivation of rhodopsin (Schertler, 2005) with a first spectral intermediate after already 200 fs.