The Ca2+ ion is one of the most important intracellular messengers in biology. In order to understand the mechanisms by which cells regulate their cytoplasmic Ca2+ concentrations, it is necessary to be able to measure those concentrations from moment to moment in living cells. Although there are now several methods of doing that, none is ideal. The Ca2+-regulated photoproteins, of which aequorin is the best-known example, have been used for this purpose for more than 25 years. These substances, which are responsible for the bioluminescence of a number of marine coelenterates, are self-contained macromolecular """"""""packages"""""""" that are readily transferred into other cells, and emit light at a rate that varies with [Ca2+]. Aequorin is the only photoprotein that is currently available in sufficient quantities to be used routinely as a Ca2+ indicator. The photoproteins would be much more widely used but for a number of features of aequorin that might well be improved through application of the methods of modern molecular biology, which can in principle: l. make rare natural photoproteins available in quantity. 2. make available photoproteins that do not occur in nature. 3. lead to the production of transgenic organisms with """"""""built in"""""""" Ca2+ indicators. Obelin is a relatively scarce photoprotein that has been reported to respond considerably faster than aequorin to rapid changes in [Ca2+]. Investigators at the Photobiology Laboratory of the Institute of Biophysics at Krasnoyarsk have recently cloned the gene for one of the isospecies of obelin, and expressed it in E. coli.
The aim of this proposal is to characterize this recombinant photoprotein as part of a wider effort to """"""""build a better aequorin"""""""". The FIRCA would support efforts in Krasnoyarsk to improve existing methods for the producing recombinant obelin, a search for genes coding different isospecies of the protein, and an effort to produce useful mutant forms of obelin by site-directed mutagenesis. The products of this work will be characterized in terms of Ca2+-sensitivity, Mg2+-sensitivity, and kinetics at Friday Harbor.
