Ultrafast time resolved protein X-ray crystallography
Lead Research Organisation:
Imperial College London
Department Name: Life Sciences
Abstract
We now have all the tools and the methodology that allows us to watch proteins function with structural resolution in real time with ultrafast time resolution, probing fundamental processes in biological activation. With the availability of femtosecond X-ray Free Electron Laser sources which now operate in the hard X-ray regime with femtosecond pulse duration, unique opportunities arise to gain fundamental new insight into ultrafast structural processes in proteins. Here, I propose a research programme on femtosecond time-resolved X-ray crystallography of light induced reactions in light sensitive protein crystals and supporting spectroscopic studies. The necessary preliminary work for such experiments are provided in the form of data collected at the Stanford Linac Coherent Light Source (LCLS), from ultrafast optical spectroscopy investigations of non-linear transitions and pulse shaping to control femtosecond population transfer performed in the home laboratory, and from supporting modeling of crystallographic data quality based on measured source parameters. In addition to the primary target, which is a femtosecond experiment with Photoactive Yellow Protein crystals, novel experiments are being developed firstly using picosecond synchrotron sources, to add to the existing choices for femtosecond pump-probe studies and focuses on photoswitchable fluorescent proteins.
Planned Impact
The principal question addressed in this proposal is fundamental in nature; it aims to reveal basic new information on the crucial early time scales of biological activation. The outcome would be of very broad and general interest, as it concerns the basic mechanisms of biological function.
There is currently a world-wide effort to 1) construct femtosecond 'hard X-ray' FEL light sources and 2) use already available machines to conduct science in new experimental regimes. Focusing on FELs that will operate in the hard X-ray regime (with Angstrom wavelength) currently LCLS and SACLA are operating and under active commissioning. The European XFEL and the SwissFEL are under construction and are expected to start user operation during the duration of this proposed project, described in the case for support. With more sources becoming available, so will opportunities to perform the proposed work and to contribute to the experimental capabilities of the end stations. This project will therefore support my ongoing involvement in this area. As stated, these will involve technical contributions to experimental stations, methods and analysis techniques, which can be transferable between the various accelerators. In addition, this support will make me well placed to contribute to future scientific case for a UK operated XFEL.
In the UK there are only a few groups who have gained access to LCLS, and JvT is the only one in the area of time resolved biomolecular structure (http://ukfel.org/wordpress/?page_id=34)
Economic Impact:
Firstly, this proposal contributes to methods to use XFELs to develop novel ways of solving protein structures, which could have significant impact on the biomedical and pharmaceutical communities as well. Further, the UK has an established past (and future) interest in this area, for which the outcome of research that uses 4th generation light sources is of direct importance. For example, the UK Structural Biology community has proposed to contribute to the SFX consortium at the European XFEL at a total cost of £16.5 M, which is currently under review at the Council. The UK currently has active accelerator development in the FEL area, particularly the CLARA and ALICE projects at Daresbury. Furthermore, STFC recently indicated that, in response to the Minister's request for 'bold and ambitious plans' the UK will 'wish to construct its own X-FEL in due course' (Prof John Womersley, 14 June 2013, Imperial College London)
People:
The delivery of highly trained personnel is evident in the combined approaches which are included in this proposal. The PDRA will use established optical techniques in the home laboratory to contribute to optimization of femtosecond photolysis of protein crystals, essential for the successful execution of ultrafast crystallography experiments. In doing so, the PDRA will be trained in the interface between femtosecond spectroscopy and femtosecond X-ray diffraction.
Society:
The novelty and importance of 4th generation light sources should be communicated to the general public, who fund these machines. JvT has an established track record engaging with the general public, engaging with popular media, and engaging with young talent, to explain and highlight the importance and impact of the fundamental work on protein dynamics. Examples of outreach by JvT are available online (and at http://www3.imperial.ac.uk/people/j.vanthor) :
Interview in Science
News Focus - Materials Science "What Shall We Do With the X-ray Laser?" By Adrian Cho
http://www.sciencemag.org/content/330/6010/1470.short
Letter from a satisfied customer to Dr Jasper van Thor
http://www.imperial.ac.uk/workspace/molecularbiosciences/Public/Outreach/outreach.docx
Catching a short-lived photoreceptor intermediate with pulsed X-rays. ESRF highlights 2011
http://www.esrf.eu/UsersAndScience/Publications/Highlights/2011/scm/scm8
There is currently a world-wide effort to 1) construct femtosecond 'hard X-ray' FEL light sources and 2) use already available machines to conduct science in new experimental regimes. Focusing on FELs that will operate in the hard X-ray regime (with Angstrom wavelength) currently LCLS and SACLA are operating and under active commissioning. The European XFEL and the SwissFEL are under construction and are expected to start user operation during the duration of this proposed project, described in the case for support. With more sources becoming available, so will opportunities to perform the proposed work and to contribute to the experimental capabilities of the end stations. This project will therefore support my ongoing involvement in this area. As stated, these will involve technical contributions to experimental stations, methods and analysis techniques, which can be transferable between the various accelerators. In addition, this support will make me well placed to contribute to future scientific case for a UK operated XFEL.
In the UK there are only a few groups who have gained access to LCLS, and JvT is the only one in the area of time resolved biomolecular structure (http://ukfel.org/wordpress/?page_id=34)
Economic Impact:
Firstly, this proposal contributes to methods to use XFELs to develop novel ways of solving protein structures, which could have significant impact on the biomedical and pharmaceutical communities as well. Further, the UK has an established past (and future) interest in this area, for which the outcome of research that uses 4th generation light sources is of direct importance. For example, the UK Structural Biology community has proposed to contribute to the SFX consortium at the European XFEL at a total cost of £16.5 M, which is currently under review at the Council. The UK currently has active accelerator development in the FEL area, particularly the CLARA and ALICE projects at Daresbury. Furthermore, STFC recently indicated that, in response to the Minister's request for 'bold and ambitious plans' the UK will 'wish to construct its own X-FEL in due course' (Prof John Womersley, 14 June 2013, Imperial College London)
People:
The delivery of highly trained personnel is evident in the combined approaches which are included in this proposal. The PDRA will use established optical techniques in the home laboratory to contribute to optimization of femtosecond photolysis of protein crystals, essential for the successful execution of ultrafast crystallography experiments. In doing so, the PDRA will be trained in the interface between femtosecond spectroscopy and femtosecond X-ray diffraction.
Society:
The novelty and importance of 4th generation light sources should be communicated to the general public, who fund these machines. JvT has an established track record engaging with the general public, engaging with popular media, and engaging with young talent, to explain and highlight the importance and impact of the fundamental work on protein dynamics. Examples of outreach by JvT are available online (and at http://www3.imperial.ac.uk/people/j.vanthor) :
Interview in Science
News Focus - Materials Science "What Shall We Do With the X-ray Laser?" By Adrian Cho
http://www.sciencemag.org/content/330/6010/1470.short
Letter from a satisfied customer to Dr Jasper van Thor
http://www.imperial.ac.uk/workspace/molecularbiosciences/Public/Outreach/outreach.docx
Catching a short-lived photoreceptor intermediate with pulsed X-rays. ESRF highlights 2011
http://www.esrf.eu/UsersAndScience/Publications/Highlights/2011/scm/scm8
Organisations
People |
ORCID iD |
| Jasper Van Thor (Principal Investigator) |
Publications
Van Thor JJ
(2015)
A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography.
in Structural dynamics (Melville, N.Y.)
Van Thor JJ
(2019)
Advances and opportunities in ultrafast X-ray crystallography and ultrafast structural optical crystallography of nuclear and electronic protein dynamics.
in Structural dynamics (Melville, N.Y.)
Sanchez-Gonzalez A
(2017)
Coincidence timing of femtosecond optical pulses in an X-ray free electron laser
in Journal of Applied Physics
Pande K
(2016)
Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein.
in Science (New York, N.Y.)
Kim TW
(2016)
Combined probes of X-ray scattering and optical spectroscopy reveal how global conformational change is temporally and spatially linked to local structural perturbation in photoactive yellow protein.
in Physical chemistry chemical physics : PCCP
Kaucikas M
(2015)
Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa.
in Proteins
Kaucikas M
(2015)
Photoisomerization and proton transfer in the forward and reverse photoswitching of the fast-switching M159T mutant of the Dronpa fluorescent protein.
in The journal of physical chemistry. B
Hutchison CDM
(2019)
Optical control, selection and analysis of population dynamics in ultrafast protein X-ray crystallography.
in Philosophical transactions. Series A, Mathematical, physical, and engineering sciences
Hutchison CDM
(2017)
X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature.
in International journal of molecular sciences
Hutchison C
(2016)
Photocycle populations with femtosecond excitation of crystalline photoactive yellow protein
in Chemical Physics Letters
| Description | We have developed and performed femtosecond time resolved X-ray crystallography experiments of light induced reactions in a photoreceptor protein Narrative impact Fastest-ever molecular imaging reveals reaction crucial for vision Press release: http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_5-5-2016-16-3-1 Scientists have tracked the reaction of a protein responding to light, paving the way for a new understanding of life's essential reactions. Every process that sustains life is carried out by proteins, but understanding how these complex molecules do their jobs depends on learning the arrangement of their atoms and how this structure changes as they react. No imaging method for observing molecular movement in such detail and speed had been available, until now. A team of biochemists and physicists, led by the University of Wisconsin-Milwaukee and Imperial College London, have documented for the first time the fundamental processes of a chemical reaction unfolding in real time. They captured images - 25 trillion per second - of a tiny crystalized protein as it reacted to light. This allowed them build up a picture of what the protein was doing every few femtoseconds, a quadrillionths of a second. The results are published today in the journal Science. Previously, scientists had relied on a method called X-ray crystallography, which could only take a static image of a protein. Now, they have been able to build up a series of crystallographic snapshots into a molecular movie over extremely short timescales. Study co-author Dr Jasper van Thor from Imperialâ?™s Department of Life Sciences said: â?œUsually, we can only image the structure after the reaction, and infer what has happened. This is the first time we have been able to image crystal structures on timescales where the proteins are still undergoing the reaction. What happens during these timescales determines the outcome of the reaction, so knowing exactly what is going on is vital. Previously our information and images of how the reactions work have been based on theory and spectroscopy. Now we can see it in reality. The team studied a yellow dye molecule at the centre of a light-sensitive protein that undergoes a shape shift as it interacts with a photon, a particle of light. The basic biological process they observed is similar to how the human eyeâ?™s retina responds to light. Using the Linac Coherent Light Source X-ray Free Electron Laser in California, the team fired intensely bright pulses at the protein, snapping images every few femtoseconds as the photon reaction progressed. Crucially, the result was enabled by careful tailoring of the visible light pulse, which is necessary with very bright and very brief pulses. Dr van Thor said: We are working in interesting regimes that are new to crystallography, where the properties of the visible pulse matter most. Next, the researchers will work on getting femtosecond details in order to actively control the dynamics. This could ultimately allow scientists to intervene in the process of protein functions by using light. Now that the technique has been proven to work, the team hope it will start to be applied across molecular biology to unravel the mechanisms of all of proteins crucial reactions for life. This puts us dramatically closer to understanding the chemistry necessary for all life, said Marius Schmidt, physics professor at UW-Milwaukee and co-author of the paper. Discovering the step-by-step process of how proteins function is necessary not only to inform treatment of disease, but also to shed light on the grand questions of biology. Imperials Dr van Thor is investigating the areas where this technology could potentially lead to new breakthroughs, and is helping develop a case for a similar instrument facility to be built in the UK. The research was carried out in collaboration with institutions from the US and Europe, including the University of Wisconsin-Milwaukee; Lawrence Livermore National Laboratory; University of Hamburg; State University of New York, Buffalo; University of Jyvaskyla; and the Max Planck Institute for Structure and Dynamics of Matter. |
| Exploitation Route | We have shown the methodology to perform femtosecond X-ray crystallography by optical control and characterisation for the first time, which is currently being published and thus provides the detailed methodology for this emerging field |
| Sectors | Energy |
| URL | http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_5-5-2016-16-3-1 |
| Description | http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_5-5-2016-16-3-1 Fastest-ever molecular imaging reveals reaction crucial for vision Press release: http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_5-5-2016-16-3-1 Scientists have tracked the reaction of a protein responding to light, paving the way for a new understanding of life's essential reactions. Every process that sustains life is carried out by proteins, but understanding how these complex molecules do their jobs depends on learning the arrangement of their atoms and how this structure changes as they react. No imaging method for observing molecular movement in such detail and speed had been available, until now. A team of biochemists and physicists, led by the University of Wisconsin-Milwaukee and Imperial College London, have documented for the first time the fundamental processes of a chemical reaction unfolding in real time. They captured images - 25 trillion per second - of a tiny crystalized protein as it reacted to light. This allowed them build up a picture of what the protein was doing every few femtoseconds, a quadrillionths of a second. The results are published today in the journal Science. Previously, scientists had relied on a method called X-ray crystallography, which could only take a static image of a protein. Now, they have been able to build up a series of crystallographic snapshots into a molecular movie over extremely short timescales. Study co-author Dr Jasper van Thor from Imperialâ?™s Department of Life Sciences said: â?œUsually, we can only image the structure after the reaction, and infer what has happened. This is the first time we have been able to image crystal structures on timescales where the proteins are still undergoing the reaction. What happens during these timescales determines the outcome of the reaction, so knowing exactly what is going on is vital. Previously our information and images of how the reactions work have been based on theory and spectroscopy. Now we can see it in reality. The team studied a yellow dye molecule at the centre of a light-sensitive protein that undergoes a shape shift as it interacts with a photon, a particle of light. The basic biological process they observed is similar to how the human eyeâ?™s retina responds to light. Using the Linac Coherent Light Source X-ray Free Electron Laser in California, the team fired intensely bright pulses at the protein, snapping images every few femtoseconds as the photon reaction progressed. Crucially, the result was enabled by careful tailoring of the visible light pulse, which is necessary with very bright and very brief pulses. Dr van Thor said: We are working in interesting regimes that are new to crystallography, where the properties of the visible pulse matter most. Next, the researchers will work on getting femtosecond details in order to actively control the dynamics. This could ultimately allow scientists to intervene in the process of protein functions by using light. Now that the technique has been proven to work, the team hope it will start to be applied across molecular biology to unravel the mechanisms of all of proteins crucial reactions for life. This puts us dramatically closer to understanding the chemistry necessary for all life, said Marius Schmidt, physics professor at UW-Milwaukee and co-author of the paper. Discovering the step-by-step process of how proteins function is necessary not only to inform treatment of disease, but also to shed light on the grand questions of biology. Imperials Dr van Thor is investigating the areas where this technology could potentially lead to new breakthroughs, and is helping develop a case for a similar instrument facility to be built in the UK. The research was carried out in collaboration with institutions from the US and Europe, including the University of Wisconsin-Milwaukee; Lawrence Livermore National Laboratory; University of Hamburg; State University of New York, Buffalo; University of Jyvaskyla; and the Max Planck Institute for Structure and Dynamics of Matter. |
| First Year Of Impact | 2015 |
| Sector | Education |
| Impact Types | Societal |