A general strategy will be developed for the creation of novel genetically encodable FRET-based sensors for small molecule secondary messengers. These sensors will be utilized in vivo to obtain molecular level information on a variety of important signal transduction pathways. Initially, the design strategy will be used to generate a new Ca2+sensor that is immune to background interactions with cellular components. Oligonucleotide directed mutagenesis will be employed to evolve a pair of altered sensing proteins (calmodulin and M13) that interact with each other in presence of Ca2+ Phage display will be used to screen large libraries of mutants, where positive and negative selections will be implemented to optimize the desired interactions. A fusion protein consisting of the two sensing proteins flanked by two fluorescent proteins will be expressed in both mammalian and bacterial cells. The boundary regions between the four proteins will be randomized using directed mutagenesis and fluorescence activated cell sorting will be used to select mutants with a high in vivo FRET response. The sorting will take place in mammalian cells and those cells exhibiting the desired fluorescent properties will be subjected to repeated rounds of sorting under increasingly stringent conditions. The successful sensors will then be used to study Ca2+dependent processes by targeting them to different cellular organelles. Ultimately, the combined use of directed evolution, phage display and FACS sorting will be extended to the development of other small molecule sensors.

F32GM067488

2003-03-01

2005-02-28