Dimitriadou, E; (2014) Redesign of calcium-regulated protein aequorin towards the development of a novel ion bioreporter. Doctoral thesis , UCL (University College London).

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Abstract

This thesis aimed to design novel sensor proteins that can identify and measure various metal ions in vivo and in situ. Metal ions play key role in the metabolism of the cell, and monitoring of calcium has helped interrogate cellular processes such as fertilisation, contraction and apoptosis. Real-time monitoring of more divalent metal ions like zinc and copper is required to gain much needed insight into brain function and associated disorders, such as Alzheimer’s and Parkinson’s disease. Aequorin is a calcium-regulated photoprotein originally isolated from the jellyfish Aequorea victoria. Due to its high sensitivity to calcium and its non-invasive nature, aequorin has been used as a real-time indicator of calcium ions in biological systems for more than forty years. The protein complex consists of the polypeptide chain apoaequorin and a tightly bound chromophore (coelenterazine). Trace amounts of calcium ions trigger conformational changes in the protein, which in turn facilitate the intermolecular oxidation of coelenterazine and concomitant production of CO2 and a flash of blue light. Aequorin’s light emitting reaction can also be triggered by a range of other divalent and trivalent cations, leading however to significantly lower light yields. Based on aequorin’s promiscuity towards other ions, this project tested the hypothesis that aequorin’s preference for certain cations could be manipulated through mutations engineered in one or more of the three calcium-binding loops (EF-hands). In order to test the hypothesis, the following six stages were performed: cloning of the apoaequorin gene for expression in E. coli; development of a high-throughput assay for expression and measurement of bioluminescent activity in microwells; design of a library containing forty eight mutant variants of aequorin; screening of the library against seven metal ions; protein purification of wild-type aequorin and one selected mutant; analysis of activity and kinetics of purified wild type and one chosen mutant against all seven ions. This work produced mutants with shifted selectivity towards new metal ions at the cost of luminescence yield. The impact of mutations is analysed and it is suggested that one of the EF-hands (EF-I) is likely to serve as a gatekeeper to aequorin’s selectivity. It was also shown that at least one mutant utilised zinc ions (that wild type failed to utilise) to achieve low levels of bioluminescent activity.

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