Moulding fluorescent proteins into biotools: engineering topology, surfaces and chromophores

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Proteins are amazing molecular machines that are essential for Life as we know it. After a century of protein research, we now have a very good understanding of how proteins are able to carry out their vast array of functions. One extraordinarily useful family of proteins, which we now understand quite well, is the family of intrinsically fluorescent proteins (FPs) that includes the green FP from Aequorea jellyfish and its numerous homologues of various color from coral. FPs have the unique (among proteins) ability to generate a visible wavelength fluorophore within their own 3-dimensional structure and thus can absorb visible light and reemit it as a red-shifted hue. In the past decade, these remarkable protein tools have enabled many exciting advances in biological research because genetically inserting a FP into a living cell or tissue allows one to 'see' biological structures that would otherwise be undetectable. FPs are credited with sparking the explosive and continuing growth in the popularity of live cell fluorescence microscopy; culminating in the awarding of 2008 Nobel Prize in Chemistry to the pioneers of FP research. My research group has worked to mould (that is, engineer) FPs into improved tools for addressing fundamental questions in life science. For example, we and others have made substantial progress in expanding the palette of FP variants into the orange and red wavelengths in recent years. However, despite this progress the vast majority of FP-based biosensors (FPs that change their fluorescence in response to specific biological events) are still based on engineered variants of Aequorea green FP. Orange and red FPs derived from coral have seen only limited use as biosensors due to the lack of variants with suitable spectral and topological properties. I propose to overcome these limitations through a combination of protein engineering and innovative biosensing strategies. Many of the potential applications of such biosensors lie in the realm of fundamental cell biology research. However, these technological advances will also be applicable to the areas of disease diagnosis and drug discovery.