Structure-Function Studies of Fluorescent Proteins

描述（由申请人提供）：绿色荧光蛋白（GFP）对分子和细胞生物学具有巨大影响，因为荧光发色团由蛋白质内的三肽序列形成，而不涉及除分子氧以外的任何辅因子。因此，GFP可以作为融合蛋白构建体引入活细胞或生物体中，以跟踪融合蛋白伴侣的定位、加工和相互作用。我们对这些荧光蛋白的兴趣有两个方面。首先，我们有一个长期的兴趣在解剖生物系统中的结构-功能关系的亲密细节。绿色荧光蛋白是一个内在迷人的系统，为我们提供了研究蛋白质基质如何控制嵌入分子的结构和性质的机会。其次，更深入地了解生色团形成的机制以及蛋白质如何调节生色团的结构和光谱特性，对于指导具有定制特性的荧光蛋白的开发以及解释当前应用的数据至关重要。还有一个强大的驱动力，对新的光敏感的玻璃纤维蛋白的发展，研究活细胞内的动态过程。 目前的建议是集中在两个假设（i）在恒定照明下发生的荧光蛋白的光发射的变化，从光诱导的生色团结构的变化，由蛋白质环境控制的结果和（ii）折叠的荧光蛋白指导和控制的生色团的形成。基于这些假设，我们将（i）确定光吸收后发色团结构的变化和（ii）确定发色团形成的机制。我们将使用拉曼光谱结合其他生物物理技术，如吸收和荧光光谱，以阐明伴随光吸收的发色团结构的变化。此外，天然化学连接将用于将非天然氨基酸插入到发色团中，以直接探测发色团形成的机制。

DESCRIPTION (provided by applicant): Green fluorescent protein (GFP) has had a huge impact on molecular and cell biology because the fluorescent chromophore is formed from a tripeptide sequence within the protein without the involvement of any cofactors other than molecular oxygen. Consequently, GFP can be introduced into live cells or organisms as a fusion protein construct in order to follow localization, processing and interactions of the fusion protein partner. Our interests in these fluorescent proteins are two fold. Firstly, we have a long-standing interest in dissecting the intimate details of structure-function relationships in biological systems. GFP is an intrinsically fascinating system that presents us with the opportunity to investigate how the protein matrix controls the structure and properties of an embedded molecule. Secondly, a deeper understanding of the mechanism of chromophore formation and of how the protein modulates the structure and spectroscopic properties of the chromophore are fundamental to guiding the development of fluorescent proteins with tailored properties and for interpreting data from current applications. There is also a strong drive towards the development of new light sensitive GFPs for studying dynamic processes inside living cells. The current proposal is centered on two hypotheses (i) that changes in light emission from the fluorescent proteins that occur under constant illumination result from light-induced changes in the chromophore structure that are controlled by the protein environment and (ii) that the folded fluorescent protein directs and controls the formation of the chromophore. Based on these hypotheses we will (i) determine the changes in chromophore structure following light absorption and (ii) determine the mechanism of chromophore formation. We will use Raman spectroscopy coupled with other biophysical techniques, such as absorption and fluorescence spectroscopy, to elucidate the changes in chromophore structure that accompany light absorption. In addition, native chemical ligation will be used to insert unnatural amino acids into the chromophore in order to directly probe the mechanism of chromophore formation.