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Elucidating structural intermediates of fluorescent proteins using X-ray free electron lasers and ultra-fast UV-visible and infrared spectroscopies – BioXFEL

Submission summary

Studying the structure and dynamics of proteins is a prerequisite for understanding their function in a cellular context. X-ray sources of a completely new kind, so called X-ray free electron lasers (XFEL), extend synchrotron-based crystallography and may eventually allow the determination of both the structure and dynamics of individual protein molecules. Today, two XFELs (LCLS, USA and SACLA, Japan) already provide the extremely intense and ultra-short X-ray pulses needed to study protein microcrystals with femtosecond (fs) time resolution. In 2016, the European XFEL (Hamburg) will start operation, offering European scientists privileged access to these new sources that are revolutionizing structural biology. The strategic aim of our proposal is to create a French Consortium and pave the way for using XFELs in structural biology, by acquiring XFEL expertise through an ambitious research project.
Our scientific aim is to investigate the hitherto unexplored ultra-fast photochemistry at play in fluorescent proteins. In particular, we propose to elucidate structural intermediates in two reversibly photoswitchable fluorescent proteins, IrisFP and rsEGFP2, at atomic resolution and in the fs time window, by making use of pump-probe XFEL-based serial femtosecond crystallography (SFX) and ultra-fast UV-visible and infrared spectroscopies. IrisFP and rsEGFP2 are crucial molecular tools to study sub-cellular structures by super-resolution microscopy techniques such as RESOLFT, non-linear SIM or PALM (Chemistry Nobel Prize 2014).
Static structures of the various dark and fluorescent states of IrisFP and rsEGFP2 have been determined by the coordinating laboratory using synchrotron X-ray crystallography. Photoswitching has been shown to involve cis-trans isomerization of the chromophore, proton transfer and rearrangements of residues in the chromophore cavity. However, mechanistic details about the sequence of molecular events remain elusive, as no crystallographic structure of a reaction intermediate has been determined so far. Our preliminary spectroscopic experiments show that photoswitching in both proteins involves transient species with characteristic time constants in the fs – ps range, too short to be structurally resolved using time-resolved Laue crystallography at a synchrotron, since that time-resolution is currently limited to 100 ps. Therefore, fs XFEL pulses are required to uncover the structural reaction intermediates. We propose to use XFELs to carry out SFX in an optical pump/X-ray probe scheme to collect diffraction patterns of light-induced reaction intermediates during IrisFP and rsEGFP2 photoswitching. Ultra-fast UV-Visible and infrared spectroscopies of protein in solution and in microcrystals will unravel the number of intermediates and their characteristic lifetimes. The structural nature of spectroscopically identified intermediates will then be identified in subsequent SFX XFEL experiments on the fs – ps and nanosecond-microsecond (ns-µs) timescales.
Preliminary experiments have substantiated the feasibility of our project. In particular, well-diffracting IrisFP and rsEGFP2 microcrystals have been produced and datasets collected at the SACLA and LCLS XFELs that allowed structure solution of the static fluorescent on state of both proteins. UV-Visible transient absorption on IrisFP and rsEGFP2 in solution provided evidence for the existence of several spectroscopic intermediate states on the fs-ps and ns-ms timescales. Our consortium has obtained beamtime for the proposed experiments at the XFELs at LCLS (# LI56; 29 April – 3 May 2015) and SACLA (#29944; 23-25 July 2015).
The proposed experiments will provide unprecedented insight into the molecular functioning of fluorescent proteins. If successful, our project will open a new field in structural biology, where the macromolecular structures of femtosecond intermediate states can be characterized at atomic resolution by using XFEL radiation.

Project coordination

Martin Weik (Institut de Biologie Structurale)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LCLS Linac Coherent Light Source
IBS Institut de Biologie Structurale
LASIR Laboratoire de Spectrochimie Infrarouge et Raman
RENNES Institut de Physique de Rennes

Help of the ANR 439,932 euros
Beginning and duration of the scientific project: November 2015 - 48 Months

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