GFP-like proteins:Diversity and determinants of color

描述（由申请人提供）：来自Aequorea victoria水母的绿色荧光蛋白（GFP）同源蛋白已成为生物体内成像不可或缺的工具，广泛应用于基础生物医学研究和药物发现。在非生物发光的珊瑚虫中发现这种蛋白质提供了一个令人兴奋的机会，可以通过提供更多的颜色来扩展这项技术。此外，一些对先进报告系统工程有价值的新特征已经成为可能，例如发射颜色的精确时间依赖性变化或光驱动荧光激活/抑制。然而，由于迄今为止只有少数有限规模的尝试从珊瑚虫中克隆gfp样蛋白质，可以预期，在对这些生物的系统调查中，可以发现更多具有新光谱和结构特征的蛋白质。特别是，这些可能包括现有蛋白质的变体，这些变体没有它们的缺点，例如寡聚化或发色团成熟缓慢。在这里，我们提出了这样一个系统的研究，旨在完整地表征珊瑚中gfp样蛋白的多样性，以便为生物医学成像应用创建大量潜在的报告。我们打算利用累积的序列/光谱特征信息来确定颜色的序列决定因素，并通过位点特异性诱变来验证它们的作用。这将极大地促进gfp类记者的合理设计。

DESCRIPTION (provided by applicant): Proteins homologous to green fluorescent protein (GFP) from the jellyfish Aequorea victoria have become indispensable tools for in vivo imaging, and are widely used in basic biomedical research and drug discovery. Finding such proteins in non-bioluminescent Anthozoa offers an exciting opportunity to expand the technology by providing more colors to choose from. In addition, a number of novel features valuable for the engineering of advanced reporter systems have become available, such as precisely time-dependent changes of emission color or light-driven fluorescence activation/inhibition. However, since only a few limited-scale attempts have been undertaken thus far to clone GFP-like proteins from Anthozoa, it can be expected that many more proteins with novel spectral and structural features can be discovered during systematic survey of these organisms. In particular, these may include variants of existing proteins that are free of their drawbacks, such as oligomerization or slow chromophore maturation. Here we propose such a systematic study aimed at complete characterization of a diversity of GFP-like proteins in Anthozoa in order to create a large inventory of potential reporters for biomedical imaging applications. We intend to use the accumulated body of information on sequence/spectral features to pinpoint sequence determinants of coloration, and to verify their role by means of site-specific mutagenesis. This would greatly facilitate rational design of GFP-like reporters.