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

蛋白质是我们所知的生命所必需的惊人的分子机器。经过世纪的蛋白质研究，我们现在对蛋白质如何能够执行其大量功能有了很好的了解。一个非常有用的蛋白质家族，我们现在已经非常了解了，是固有荧光蛋白（FP）家族，包括来自水母的绿色FP和来自珊瑚的各种颜色的许多同系物。FP具有独特的（在蛋白质中）在其自身的三维结构内产生可见波长荧光团的能力，因此可以吸收可见光并将其重新发射为红移色调。在过去的十年中，这些非凡的蛋白质工具使生物研究取得了许多令人兴奋的进展，因为将FP基因插入活细胞或组织中可以让人们“看到”否则无法检测到的生物结构。FP被认为引发了活细胞荧光显微镜普及的爆炸性和持续增长;最终将2008年诺贝尔化学奖授予FP研究的先驱。 我的研究小组一直致力于将FP塑造（即工程师）为解决生命科学中基本问题的改进工具。例如，近年来，我们和其他人在将FP变体的调色板扩展到橙子和红色波长方面取得了实质性进展。然而，尽管取得了这一进展，但绝大多数基于FP的生物传感器（响应于特定生物事件而改变其荧光的FP）仍然基于水母绿色FP的工程化变体。来自珊瑚的橙子和红色FP由于缺乏具有合适光谱和拓扑性质的变体而仅被有限地用作生物传感器。我建议通过结合蛋白质工程和创新的生物传感策略来克服这些限制。这种生物传感器的许多潜在应用在于基础细胞生物学研究领域。然而，这些技术进步也将适用于疾病诊断和药物发现领域。

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.