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

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.

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.