Femtosecond to Millisecond Photo-dynamics of Third Generation Fluorescent Proteins

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Optical microscopy has been central to biology for centuries. In recent decades fluorescence microscopy has developed as an exceptionally sensitive and universally employed tool in the life sciences. Its power was dramatically enhanced in the 1990s through the discovery of the green fluorescent protein (GFP). This allowed a fluorescent label (GFP) to be irreversibly and selectively expressed attached to a specific target protein, permitting the location, dynamics and function of that protein to be probed in a living cell. More recently, photoswitchable fluorescent proteins were discovered. These fluorescent proteins can be reversibly switched between fluorescent (on) and nonfluorescent (off) states by light. This property allowed the development of super-resolution fluorescence bioimaging, which improved the spatial resolution of fluorescence microscopy by more than 10 times, allowing details much smaller than the wavelength of light to be observed in living systems. However, applications of photoswitchable proteins are limited because the light used to generate fluorescence from their 'on' states also switches the proteins 'off', causing the image to fade. A higher level of control is offered by the recently discovered third generation fluorescent proteins (3G FPs), which have three states, an off-state and a switching state in thermal equilibrium with an on-state. These three states can be independently excited, so switching off is decoupled from observation from the on-state. This development will lead to enhanced super-resolution imaging, and has the potential for the development of new multicolour imaging methods. However, the mechanism connecting these states is completely unknown.Our experiments will allow us to probe in detail the effects of light on all three states of 3G FPs. We will measure the rates of the interconversion between the three states as well as the structural changes that accompany them. Since the fastest of the interconversion reactions are extremely fast (thousand-billionths of a second) we will use the tools of ultrafast laser spectroscopy to make our observations. Our ultrafast experiments probe populations of reactant, intermediate and product states through their absorption spectroscopy, while their structures are probed through ultrafast vibrational spectroscopy. These measurements will lead to a detailed picture of the photoconversion mechanism in 3G FPs. Once the mechanism is established, we will apply the tools of chemical biology to make mutations in key residues that will (i) test our ideas of the mechanism and (ii) optimise the photoswitching rate, and thus yield superior 3G FPs for bioimaging.Photoactive proteins such as FPs offer a unique opportunity to observe protein structure evolution in real time. In particular time resolved vibrational spectroscopy yields structural data from femtoseconds out to milliseconds. This information has great importance, as it can be compared with the results of computational calculations and independent experiments on protein structural dynamics. Together this will yield the most detailed insights yet into the dynamics of proteins undergoing their function, which in turn enhances our understanding of the nature of drug-protein interactions.