Photoelectron spectroscopy in a liquid microjet: unravelling the excited state dynamics of photoactive proteins

The extensive use of efficient light-induced processes in nature is inspiring efforts to exploit similar processes in functional synthetic systems. For example, fluorescent proteins have revolutionalised biological imaging. However, our understanding of the crucial role of the protein surrounding the chromophore in photoactive proteins is still far from complete. The aim of this research is to gain a molecular level understanding of the interactions between photoactive protein chromophores and their environment by a systematic investigation of the electronic structure and excited state dynamics of a series of chromophores in vacuo, in solution and in protein.

Photoelectron spectroscopy is a particularly valuable tool for measuring the binding energies of electrons in molecules and femtosecond time-resolved photoelectron spectroscopy has emerged as a very powerful technique for probing the flow of energy in a molecule following photoexcitation. In femtosecond time-resolved photoelectron spectroscopy, a femtosecond laser pulse (pump) excites a molecule and after some delay a second femtosecond laser pulse (probe) ionises the molecule. The kinetic energy and angular distribution of the resulting photoelectrons provides information about the electronic and vibrational states of the molecule at the time of ionisation. Recording a series of photoelectron spectra at different pump-probe delays allows us to record a molecular "movie" of the flow of electronic and vibrational energy in the molecule. Time-resolved photoelectron spectroscopy has proved remarkably successful for investigating electronic structure and dynamics in the gas phase and in solids but aqueous solutions have presented more of a challenge. Recent technical advances in liquid microjet technology have enabled time-resolved photoelectron spectroscopy in solutions to become a real and exciting possibility. We will exploit these developments to create a photoelectron spectroscopy apparatus that is capable of investigating the femtosecond dynamics of chromophores in solution and in their protein environments.

In a biological system, the molecular dynamics after photoexcitation are controlled by the molecular and electronic structure of the chromophore and by its interaction with the environment. For example, the environment of the GFP chromophore defines its optical properties: the chromophore is strongly fluorescent inside its barrel-shaped protein, while the fuorescence is lost when the protein is denatured but it returns upon renaturation; the isolated chromophore is non-fluorescent in aqueous solution and it is also non-fluorescent in the gas phase, yet the absorption spectrum of the isolated molecule in the gas phase is remarkably similar to that in the protein. In order to unravel the important role of the environment in defining the optical properties of fluorescent proteins, we will investigate how systematic changes to the electronic and structural properties of the chromophore and mutations to the protein influence binding energies and electronic relaxation following photoexcitation. High-level electronic structure and dynamics calculations will assist the interpretation of the experimental results. Organic chemistry and molecular biology methods will be employed to create the series of chromophores and proteins for systematic evaluation of the influence of electronic, structural, and environmental changes.

The multidisciplinary team that has been assembled is ideally suited to tackle this important problem which will have an impact in many areas of science.

The proposed work is fundamental research that will have direct impact on knowledge generation, people and society. It also underpins a range of actual and emerging technologies and will have long term influence on the development of devices that rely on light-matter interaction.

Knowledge generation:
The most significant output of this research will be the creation of a unique combined experimental and theoretical toolkit for understanding how photoactive proteins work and how nature has evolved efficient ways of doing things. Specifically, by the end of the proposal, this capability will have been demonstrated through a detailed investigation of the electronic structure and dynamics of the green fluorescent protein and variants, a prototypical family of fluorescent proteins that have revolutionised the life sciences. The toolkit will not be limited to these systems and will be used in the future to study other chromophores of scientific interest in many areas and to detail the role of the surrounding solvent or protein environment. The multidisciplinary nature of the science and its potential applications in the life sciences will ensure high impact publications in journals aimed at general audiences.

People:
Training the next generation of scientists is important for sustainable science. Three PDRAs and at least two PhDs will be trained in state-of-the-art physical chemistry, computational chemistry and synthetic organic chemistry and biology. Training activities for the PDRAs will focus on enhancing their interdisciplinary skills and building a research team with a shared understanding of project goals. This will be achieved by day-to-day training and attending courses in relevant areas. In addition to training our PDRAs, the impact from our work will be maximised by training other researchers to use our toolkit. For example, Graham Worth (GAW) is involved in workshops to teach researchers how to use his software to study photoexcited molecular dynamics.

Society:
The investigators are committed to, and engaged in, communicating scientific strategies and discoveries to the public and between them have given lectures and demonstrations at the Royal Institution, the Edinburgh Science Festival, Disneyland and a number of schools. During the course of this project, one of our aims will be to develop a new experiment for use in outreach talks to demonstrate how efficient light-induced processes in nature are inspiring efforts to exploit similar processes in functional synthetic systems - in this case photoactive proteins. In addition to providing a new resource for outreach activities aimed at schools, another aim will be to prepare a display to showcase our science at a Royal Society Summer Science Exhibition.

Economy:
In terms of the long-term impact of this work, the potential gains are expected to be hugely significant to science and society. Light-matter interactions are becoming increasingly important in a number of technologies crucial for the long-term sustainability of society. Perhaps the most obvious is the need for clean energy sources in which solar energy is a key - a global grand challenge. Developing a molecular level understanding of light-matter interactions is also crucial for emerging advances in healthcare technologies such as in vivo imaging and various phototherapies. Other significant applications include information technologies in the form of optical switches and optical storage devices. We will hold a networking meeting towards the end of the second year of the project to forge links with interested industrial partners and to investigate possibilities for new collaborations with end users of both the results of the research and the hardware and software developed during the project.