Structural Studies on Aequorin

本研究的目的是了解钙活化发光蛋白aequorin的作用机制。这项工作将集中在x射线晶体学的结构测定上，以在原子水平上识别蛋白质的成分和使水芹蛋白生物发光的显色配体——铜肠嗪。一旦确定了天然无钙全蛋白的结构，其他形式的全蛋白的结构将被研究。这些包括钙活化后的水麒麟蛋白（蓝色荧光蛋白），含有修饰形式的发色团的水麒麟蛋白，以及带有蛋白质突变的水麒麟蛋白，这些突变旨在确定哪些残基参与了反应机制以及如何参与反应机制。在再生过程中，蓝色荧光蛋白可以转换回活性绿蛋白，其机制也将被研究。除了了解这些过程的机制外，这些研究还将旨在通过蛋白质突变或发色团的改变来产生修饰形式的黄蜡蛋白，以产生具有不同离子选择性，响应性和发射波长的形式。生物发光是一种化学能量被生物分子转化为光的过程。许多动物使用这种分子来传递信号；对彼此，对敌人，对猎物。一种这样的分子是蛋白aequorin。Aequorin已被证明具有特殊的科学价值，因为它会对钙的存在发出光。尽管在实验室中广泛使用了水芹素，但当加入钙时，它将化学能转化为光能的机理的细节仍然知之甚少。这项工作旨在通过使用x射线晶体学来确定aequorin单个原子的位置以及它们如何与钙相互作用以产生光，从而提供对这些细节的理解。

The objective of this research is to understand the mechanism of action of the calcium-activated light-emitting protein aequorin. The work will focus on structure determination by X-ray crystallography to identify, at the atomic level, the components of the protein and the chromophoric ligand, coelenterazine, which enable aequorin bioluminescence. Once the structure of the native, calcium-free, holoprotein is determined, the structures of other forms will be studied. These include aequorin after calcium-activation (blue fluorescent protein), aequorin containing modified forms of the chromophore, and aequorin with protein mutations which are designed to establish which residues are involved in the reaction mechanism and how. The mechanism(s) involved in the regeneration, in which the blue fluorescent protein can be converted back to active aequorin, will also be investigated. In addition to understanding the mechanisms of these processes, these studies will also be aimed at producing modified forms of aequorin, by either mutation of the protein or changes in the chromophore, to produce forms with different ion selectivity, responsiveness and emission wavelength.Bioluminescence is a process whereby chemical energy is converted into light by a biological molecule. Many animals use such molecules for signaling; to one another, to enemies or to prey. One such molecule is the protein aequorin. Aequorin has proved of particular scientific value because it emits light in response to the presence of calcium. Despite the extensive laboratory use of aequorin, the details of the mechanism by which it converts chemical energy to light when calcium is added remains poorly understood. This work aims to provide an understanding these details by using x-ray crystallography to determine the position of the individual atoms of aequorin and how they interact with calcium to generate light.