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产生了巨大的影响，因为荧光发色团是由蛋白质内的三肽序列形成的，除了分子氧之外没有任何辅助因子的参与。因此，GFP可以作为融合蛋白构建物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，我们对解剖生物系统中结构-功能关系的亲密细节有着长期的兴趣。GFP是一个本质上迷人的系统，它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，更深入地了解发色团的形成机制以及蛋白质如何调节发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中的数据至关重要。开发新的光敏gfp用于研究活细胞内的动态过程也是一个强有力的推动力。

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.