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

蛋白质是令人惊叹的分子机器，对我们所知的生命是必不可少的。经过一个世纪的蛋白质研究，我们现在对蛋白质如何能够执行其广泛的功能有了很好的理解。一个非常有用的蛋白质家族，我们现在已经很好地了解了，是固有荧光蛋白(FP)家族，包括来自Aequorea水母的绿色FP及其来自珊瑚的大量不同颜色的同源物。FPS在蛋白质中具有独特的能力，可以在自己的三维结构中产生可见波长的荧光团，从而能够吸收可见光并将其重新发射为红移的色调。在过去的十年里，这些非凡的蛋白质工具在生物学研究中取得了许多令人兴奋的进展，因为通过将FP基因插入活细胞或组织中，人们可以‘看到’原本无法检测到的生物结构。FPS被认为是引发活细胞荧光显微镜普及的爆炸性和持续增长的原因；最终将2008年诺贝尔化学奖授予FPS研究的先驱。我的研究小组致力于将FP塑造(即设计)为改进的工具，以解决生命科学中的基本问题。例如，近年来，我们和其他人在将FP变体的调色板扩展到橙色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，绝大多数基于FP的生物传感器(根据特定生物事件改变其荧光的FP)仍然基于Aequorea green FP的工程变体。由于缺乏具有合适光谱和拓扑性质的变体，从珊瑚中提取的橙色和红色FP作为生物传感器的应用有限。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存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.