International Collaboration in Chemistry on Control of Excited State Proton Transfer in GFP

Lead Research Organisation: Imperial College London
Department Name: Life Sciences


The 2008 Nobel Prize recognized the revolutionary biological applications of the green fluorescent protein (GFP) and related fluorescent proteins as tools for monitoring protein localization and trafficking in cells and tissues. However, the prototype GFP, from the jellyfish Aequorea victoria, is also unique in exhibiting highly efficient (>80%) proton transfer following absorption of a photon. Proton transfer contributes to most biological reactions, but typically takes place on time scales that are difficult to observe directly. The ability to trigger proton transfer with a femtosecond light pulse thus yields unequalled opportunities for understanding how proteins control this ubiquitous process. Our proposed investigations proposal is hypothesis-driven and aims to understand the dynamics and control of the ultrafast excited state proton transfer (ESPT) reaction. In particular, we hope to identify low frequency promoting vibrations that form an essential element of theoretical treatments of proton transfer. We present preliminary measurements of low frequency vibrational coherences that occur within the duration of the ESPT reaction. Control of ESPT in GFP is currently fervidly discussed and we propose to help resolve outstanding issues such as the enthalpic and entropic reaction barriers, the role of chromophore cooling, low frequency mode identification and assignment, and heterogeneity and interconversions of the protein-chromophore structure. Ultrafast kinetics, vibrational coherence spectroscopy (VCS), transient visible-visible and visible-infrared absorption spectroscopy, femtosecond stimulated Raman spectroscopy (FSRS), and coherent control spectroscopy will be used to dissect the microscopic details. Particular emphasis will be placed on temperature dependent studies. Further development of high level computational chemistry calculations that include the protein environment will aid in interpretation of the spectroscopic results. In turn, the experimental results will provide an essential calibration for the computational methods.

Planned Impact

This project has broad impact on the field of protein dynamics and focuses on proton transfer within the GFP protein system. Studies of such interactions are crucial to our basic understanding of living systems. They also have a health-related significance to society at large, since a fundamental understanding of biomolecules at the molecular level will ultimately enhance our ability to diagnose and treat disease states that appear at the macroscopic level. Students (both graduate and undergraduate) are trained in cutting edge optical and biological techniques that benefit society through the enhancement of the human resource infrastructure of the academic, medical, biotechnology, and photonics industries. Instrumentation and theoretical development takes place under the sponsorship of this proposal, which enhances the infrastructure for laser based research on living state materials at the molecular level. This offers the potential for applications and economic development in a variety of medical sub-fields, from imaging and diagnosis to new clinical treatments. The knowledge obtained is disseminated through lectures, seminars, and published papers. Outreach to underrepresented groups and the general public also takes place as a result of this project as described in the broader impact section of the NSF proposal. The UK and US teams field requests from journalists to help them understand scientific ideas and technology developments in order to benefit the general public. The US team has also been actively working to educate the general public about scientific issues, and one of the PIs has served on the Board of Directors of the Telluride Science Research Center (TSRC). TSRC works to help disseminate scientific knowledge to other scientists and to the general public and the PI has organized TSRC public lectures and presented a large outreach presentation to kids (and their parents) on the topic What is Light? . In the UK, Jasper van Thor holds a Royal Society University Research Fellowship. The Royal Society, which is based in London, is very active in informing the general public on science issues that impact aspects of society. The world's oldest science academy offers many platforms for the public, politicians, and scientists to discuss and inform on global and local issues. As a Research Fellow JvT has, for example, contributed to information gathering and also regularly attends meetings at the Society. In the UK, the Royal Society has a unique position to support scientific research and individual researchers, and is able to use this influence to take part in important political and social processes.


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Fare C (2020) Radical-Triggered Reaction Mechanism of the Green-to-Red Photoconversion of EosFP. in The journal of physical chemistry. B

Description Tenboer J., Basu S., Zatsepin N., Pande K., Frank M., Hunter M., Milathianaki D., Boutet S., Williams G., Oberthuer D., Heymann M., Kupitz C., Conrad C., Coe J., Roy-Chowdhury S., Weierstall U., James D., Wang D., DePonte D., Barty A., White T., Yefanov O., Segelke B., Gati C., Seuring C., Srajer V., Henning R., Grant T., Schwander P., Fromme R., Ourmazd A., Moffat K., Van Thor J J., Spence J., Fromme P., Chapman H., Schmidt M.. Time Resolved Serial Femtosecond Crystallography Captures High Resolution Intermediates of Photoactive Yellow Protein. (2014) Science. In Press Summary: This collaborative work describes for the first time a high resolution X-ray structural observation of a photointermediate in a protein crystal using an X-ray free electron laser. This work lays the technical foundations for ultrafast femtosecond time resolved pump-probe crystallography. Kaucikas, M., Tros, M. and van Thor, J.J. Photoisomerisation and proton transfer in the forward and reverse photoswitching of the fast-switching M159T mutant of the Dronpa fluorescent protein. (2014) Journal of Physical Chemistry B 10.1021/jp506640q Summary: We extend time resolved infrared measurements of the reversibly photoswitchable fluorescent protein (previously reported for the wild type in Nature Comm., 2013) to include a fast switching mutant. We observe for the first time meta-stable intermediates in the photoswitching pathways in both directions. Jasper J. van Thor*, Mark M. Warren, Craig N. Lincoln, Matthieu Chollet, Henrik Till Lemke, David Mark Fritz, Marius Schmidt, Jason Tenboer, Zhong Ren, Vukica Srajer, Keith Moffat, Tim Graber (2014) Signal to noise considerations for single crystal femtosecond time resolved crystallography of the Photoactive Yellow Protein Faraday discussions 171, DOI: 10.1039/C4FD00011K Summary: This work presents for the first time the application of the rotation method with monochromatic radiation at 9 keV from an XFEL (LCLS) for femtosecond time resolved protein crystallography. The measurements are evaluated in terms of the crystallographic data quality which is dominated by source noise characteristics. One important discovery is the contribution of mode distortion and mode noise on the signal to noise of the structure factor amplitude determination. A model is developed that propagates experimentally measured noise parameters to predict crystallographic R values from the known mosaic spread and particularly the sensitivity with which small structure factor amplitude differences measure femtosecond population transfer Lee M. Thompson, Aurélie Lasoroski, Paul M. Champion, J. Timothy Sage, Michael J. Frisch Jasper J. van Thor* ,Michael J. Bearpark* (2014) Analytical Harmonic Vibrational Frequencies for the Green Fluorescent Protein Computed with ONIOM: Chromophore Mode Character and Its Response to Environment. J Chem Theo Comp. Vol:10, ISSN:1549-9618, Pages:751-766 Summary: This is a benchmark study, in collaboration with Gaussian Inc, which performs analytical second order derivative calculations in the ONIOM scheme which includes the explicit protein environment of the chromophore. Extensive comparisons with experimental data are made on the basis of mode character analysis. Mark. M. Warren, Marius Kaucikas, Ann Fitzpatrick, Paul Champion, J. Timothy, Sage, Jasper J. van Thor (2013) Ground state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa. Nature Communications 4:1461 doi: 10.1038/ncomms2460 Summary: The reversible photoswitching between the 'On' and 'Off' states of the fluorescent protein Dronpa involves photoisomerisation as well as protein sidechain rearrangements, but the process of interconversion remains poorly characterized. Time-resolved infrared measurements presented here monitor the sequence not only of these structural changes but also of proton transfer events, which are crucial to the development of spectroscopic contrast essential for practical applications such as super-resolution imaging. Light induced deprotonation of the chromophore phenolic oxygen in the 'Off' state is a thermal ground state process which follows ultrafast (9ps) trans-cis photoisomerisation, and so does not involve Excited State Proton Transfer (ESPT). Steady state infrared difference measurements definitively excludes protonation of the imidazolinone nitrogen in both the 'On' and 'Off' states, which are thus identified to have the anionic cis and neutral trans chromophore, respectively. Ultrafast pump-probe infrared measurements of the 'On' state reveals a weakening of the hydrogen bonding between Arg 66 and the chromophore C=O, which could be central to initiating structural rearrangement of Arg 66 and His 193 coinciding with the low quantum yield cis-trans photoisomerisation Lincoln C.N., Fitzpatrick AE, van Thor JJ (2012) Photoisomerisation Quantum Yield and Non-linear Cross-Sections with Femtosecond Excitation of the Photoactive Yellow Protein. Phys Chem Chem Phys, 14, 15752-15764 Summary: This work demonstrates femtosecond spectroscopy for the purpose of establishing and optimising ultrafast pump-probe XFEL experiments. We have conducted an extensive femtosecond spectroscopy investigation of the ultrafast photochemical dynamics of the Photoactive Yellow Protein (PYP) photoreceptor, in order to optimise the time resolution and photoproduct yield for pump-probe femtosecond X-ray crystallography. It is found that photochemical dynamics are a major limitation to the achievable time resolution for femtosecond pump-probe experiments. From systematic power density titration and addition of second order dispersion, all non-linear cross sections were extracted from solving the population transfer equations for the complex multi-level scheme that describe the multiple femtosecond photochemical pathways of PYP. Whereas off-resonance conditions reveal either limitations to photoproduct formation (photoisomerisation) from either two photon absorption (blue edge) or stimulated emission cross section (red edge), optimal conditions are directly on resonance at 450nm with addition of chirp that matches the ~300 fs time constant dynamic Stokes shift and so suppresses the stimulated emission cross section. We show that under these conditions intense pulses propagate deep into optically dense materials (crystals) achieving photoisomerisation yield (~20%) that approach the primary quantum yield. We applied these exact conditions at the XPP beamline in April 2012 for pump-probe experiments with PYP crystals. Jasper J. van Thor*, Craig N. Lincoln, Brendon Kellner, Konstantinos N. Bourdakos, Lee Thompson, Mike Bearpark, Paul M. Champion, J. Timothy Sage* (2012) Ultrafast vibrational dynamics of parallel excited state proton transfer reactions in the Green Fluorescent Protein. Vib. Spec. 62 (2012) 1-6 Summary: This work is an overview of a number of technical issues that have decided issues of vibrational mode assignment. This includes a numerical procedure to derive dipole gradients from computation of molecular dipoles with displacement in the normal modes.
Exploitation Route A key result of our work is that we have established technical requirements for femtosecond photolysis of protein crystals using pulse shaping techniques, and additionally we have developed method for sensitive detection by X-ray crystallography using novel XFEL sources. Furthermore, we have contributed fundamental knowledge on the photowitching reactions of fluorescent proteins that are very important for biological fluorescence microscopy applications
Sectors Other

Title Ultrafast spectroscopy modelling toolbox 
Description USER-FRIENDLY DATA ANALYSIS FOR MULTI-PULSE SPECTROSCOPY User friendly Graphical User Interface, Matlab based Global analysis of time dependent data Demos and manual supplied with the program Singular Value Decomposition (SVD) and fitting of Left Singular Vectors using any model Instrument Response Function (IRF) and chirp Standard library of reaction models supplied Graphically design any connectivity ('target') model Store and re-use fitting session parameters PHOTOSELECTION THEORY APPLIED TO MULTIPLE PULSES IN THE PRESENCE OF DECAY Photoselection theory for finite bleach applied to pump-probe and multi-pulse (pump-dump-probe and pump-repump-probe) spectroscopy Explicit population, laser field orientation and decay dependence Angle resolved and ensemble averaged photolysed fraction calculation Beam diameters included for pump1, pump2 and probe Choice of Gaussian and multi-mode beams Fractional photolysis calculation required for correcting pump-dump-probe signal amplitudes Provides power density dependent corrections to measured anisotropy in photoselection studies THIS SOFTWARE IS FREE TO DOWNLOAD AND USE. Please cite: Modelling Multi-Pulse Population Dynamics from Ultrafast Spectroscopy. van Wilderen, L.J.G., Lincoln, C.N. & van Thor, J.J. (2011) PLoS ONE 6(3): e17373. doi:10.1371/journal.pone.0017373 email: 
Type Of Technology Physical Model/Kit 
Year Produced 2011 
Impact This software has been taken up widely in the ultrafast spectroscopy community