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

将荧光蛋白模制成生物工具：工程拓扑、表面和发色团

• 批准号: 288338-2010
• 负责人: Campbell, Robert
• 金额: $ 5.46万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=571397
• 关键词: Moulding; fluorescent; proteins; into; biotools

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.

蛋白质是令人惊奇的分子机器，对我们所知的生命至关重要。经过一个世纪的蛋白质研究，我们现在对蛋白质如何执行其广泛的功能有了很好的了解。我们现在已经非常了解的一个非常有用的蛋白质家族是固有荧光蛋白 (FP) 家族，其中包括来自水母水母的绿色 FP 及其来自珊瑚的各种颜色的众多同源物。 FP 具有在其自身 3 维结构内产生可见波长荧光团的独特能力（在蛋白质中），因此可以吸收可见光并将其以红移色调重新发射。在过去的十年中，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，因为通过基因方式将 FP 插入活细胞或组织中可以让人们“看到”原本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的爆炸性和持续的普及；最终将 2008 年诺贝尔化学奖授予了 FP 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（响应特定生物事件而改变荧光的 FP）仍然基于水母绿色 FP 的工程变体。由于缺乏具有合适光谱和拓扑特性的变体，源自珊瑚的橙色和红色 FP 作为生物传感器的用途有限。我建议通过蛋白质工程和创新生物传感策略的结合来克服这些限制。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。