Structural Studies on Aequorin

本研究的目的是了解钙激活发光蛋白aequorin的作用机制。这项工作将集中于通过X射线结晶学的结构确定，以在原子水平上确定蛋白质和发色配体coelenterazine的组成，这些配基能够使Aequorin生物发光。一旦确定了天然的、无钙的全蛋白的结构，就会研究其他形式的结构。其中包括钙激活后的Aequorin(蓝色荧光蛋白)，含有发色团修饰形式的Aequorin，以及具有蛋白质突变的Aequorin，旨在确定哪些残基参与了反应机制以及如何参与。参与再生的机制(S)也将被研究，在这个再生过程中，蓝色荧光蛋白可以转化回活性的aequorin。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的突变或生色团的变化来产生修饰形式的水飞蓟素，以产生具有不同离子选择性、响应性和发射波长的形式。生物发光是生物分子将化学能转化为光的过程。许多动物使用这种分子来传递信号；彼此之间、敌人之间或猎物之间。其中一个这样的分子是蛋白质aequorin。Aequorin已被证明具有特殊的科学价值，因为它在存在钙的情况下会发光。尽管木犀草素在实验室中得到了广泛的应用，但人们对它在添加钙时将化学能转化为光的机制仍知之甚少。这项工作旨在通过使用x射线结晶学来确定aequorin的各个原子的位置以及它们如何与钙相互作用来产生光来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.