This project employs a combined experimental and computational approach to develop mechanisms by which ordinary proteins can be converted into molecular switches. Induced domain swapping (INDOS) is a technology that allows the function of a protein or enzyme to be switched on and off by a small molecule drug, metal ion, or change in pH. INDOS will be used to manipulate cellular pathways, in living cells and in real time, to examine their roles in health and disease. Protein fragment exchange (FREX) and alternate frame folding (AFF) are mechanisms by which an arbitrary binding protein can be turned into a fluorescent biosensor. We will generate a family of sensors for detecting a variety of biological targets in vivo and in vitro. The impact of this study lies in that the switching mechanisms being developed: (i) are general and can be applied to many proteins; (ii) are versatile, allowing the resulting switches to perform a variety of new and useful functions; and (iii) will provide insight into their underlying molecular phenomena (domain swapping, fragment complementation, and circular permutation), all of which occur commonly in nature yet are poorly understood.