Serial Femtosecond Crystallography of Optogenetic Function

The ability to design synthetic light-sensitive materials that can be genetically encoded provides biologists the means and opportunity to sense and control biological function and environment. In order to learn how these materials function it has very recently become possible to make very fast 'snapshots' of the light-induced motions using X-ray crystallography, which is even possible for very short time scales that still involves the excited electrons (on femtosecond time scale). Novel 4th generation light sources can now be used to record such 'molecular movies' which is a breakthrough technology that we only very recently demonstrated. In the very near future we anticipate being able to record even better quality movies, as the European XFEL in Hamburg that allows much more data to be recorded will start user operation.
Where previously we could only use laser spectroscopy techniques we can now actually 'watch' the very first motions of proteins directly after activation. It is known that the 'outcome' of biological reactions involving either sensing or activation (via 'actuation') is determined in these very first motions that occur on time scales typically less than picoseconds. For example, in the case of a bi-directional photochromic fluorescent protein the efficiency of switching in one direction is excellent at 30%, while the efficiency of switching in the reverse direction is poor at less than 0.05%, potentially limiting optogenetics applications. We are thus in a position to ask the question what determines this dramatic difference in the outcome of the reactions, and provide structural and dynamical information that will be highly valuable for feeding back for rational optogenetics design.

The objective of this proposal is to develop and execute a set of new time resolve X-ray crystallography experiments using the specific technique of Serial Femtosecond Crystallography, in order to follow the ultrafast motions of optogenetics materials with biological importance. Combined with femtosecond optical excitation and cross-correlation of arrival time (time stamping) data binning allows the reconstruction of femtosecond time resolved 'snapshot' pump-probe datasets that together form molecular movies. Everything is in place to proceed with newly identified targets that are selected from the optogenetics field on the basis of their biological significance and impact.The M13-cpGFP-CaM chimera, GEM-GECO1, is a synthetic fusion including a fluorescent protein, Calmodulin and an M13 helical domain that confers an optical readout of the cellular Ca2+ concentration which is an important physiological messenger. Published ultrafast Raman spectroscopy has shown time scales and motions associated with protein vibrations as well as the proton transfer that affects the measured steady state Stokes Shift, are excellent targets for femtosecond time resolved pump-probe TR-SFX measurements. We will develop suitable microcrystals of the construct, using seeded batch-crystallisation techniques that are also successful for photochromic fluorescent proteins or alternatively search for conditions using the liquid handling robots at Imperial. We will make the necessary optical measurements of the linear and non-linear multi-photon transformation in crystalline samples, using methods and instrumentation previously reported and used for recent successful TR-SFX of the photoactive yellow protein. These will establish the suitable optical parameters for pumping the microcrystals at the XFEL beamlines. At the Eu-XFEL, an X-ray crystallographic equivalent of an impulsive Raman spectroscopy experiment is envisioned, which retrieves frequency resolved high bandwidth vibrational coherence

Economy

This proposal concerns the relatively fundamental research on the development of novel XFEL based measurements of optogenetics materials. The research addresses BBSRC the two priority areas in Synthetic biology (optogenetics) as well as in Technology developments in the biosciences (TR-SFX). Designing and modifying optogenetics materials is a key area in synthetic biology as it allows Engineering cells/organisms to include systems or parts not found in nature to impart new capacities or chemistry. For technology developments this proposal demonstrates strong multidisciplinary partnerships between bioscientists and researchers in the physical sciences, engineering and information technology disciplines.

The case for national economic importance is made two-fold. Firstly, the research is proven high-impact and at the forefront of XFEL science. This work will thus strengthen the UK competitiveness internationally. Secondly, the successful outcomes of early science in the first few years of user operation of the currently operating hard X-ray FELs (LCLS and SACLA) contributes to the UK scientific case for a national facility that is currently being developed (ukfel.org).

People
The multidisciplinary work involves researchers from various backgrounds, and collaborations at XFEL stations with scientists and engineers that provide excellent training opportunities for PDRAs and participating students and collaborators. The PI has established collaboration with many groups and people working in the XFEL and ultrafast spectroscopy fields. JvT collaborates with Anders Madsen at the European XFEL MID beamline (see van Thor and Madsen, 2015, Structural dynamics), which provides further opportunities for collaborative and developmental activities involving instrumentation development and theory. Other collaborations and associations are with the UK XFEL Hub (Allen Orville), Diamond-Imperial (Isabel Moraes and So Iwata), RIKEN (So Iwata), Argonne/APS BioCARS (Keith Moffat), the BioXFEL consortium (Petra Fromme, John Spence) and others. This provides an environment for participants in this research which is excellent and at the forefront of the field.


Knowledge and Outreach

The knowledge generated by the proposed experiments will be communicated through the standard routes: research papers and conference presentation, but in addition also press releases and outreach. The home page (http://www.imperial.ac.uk/people/j.vanthor) shows a link to a news item that describes our recent TR-SFX publication in Science.
http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_5-5-2016-16-3-1
The PI has a strong track record in publishing in top tier journals, and is a frequent presenter at international conferences (http://www.imperial.ac.uk/people/j.vanthor/research.html)
In addition, the PI has organised international conferences, in London and Telluride (the TSRC conference on Protein Dynamics).