Structural Studies on Aequorin

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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.