Abstract:

The development of efficient tools for controlling protein function in living cells is one of the greatest challenges of the proteomic era. Traditionally, genetic approaches such as transcriptional regulation and gene knockouts have been used to control gene function in a highly specific and generalizable manner, but these strategies suffer from transcriptional and translational delays before a functional product is formed. Chemical genetics on the other hand utilize small molecules to dosably activate or inactivate a particular protein target, but they require the discovery of a different small-molecule regulator for every individual protein target. In this work, engineered protein-based molecular switches have been introduced as novel tools for regulating protein activity that combine the advantages of genetic and chemical genetic approaches. The first part of this dissertation describes the development of a small-molecule-controlled protein self-splicing element (intein). This engineered intein can be used to inactivate arbitrary target proteins by genetic insertion and then reactivate them post-translationally when the splicing reaction is activated and intein self-excision takes place. Protein activation in vivo can be achieved by simple addition of the appropriate small-molecule compound in the growth medium, with a fairly rapid response and in a dose-dependent manner that allows precise specification of the levels of active protein in the cell. In the second part of this thesis, it is demonstrated that protein-based molecular switches can be used for the construction of simple biosensors. By creating engineered chimeric proteins that couple ligand binding to a pharmaceutically important hormone receptor with the activity of a reporter protein that is required for cell growth, engineered bacterial strains have been generated whose survival relies on the presence of compounds with hormone-like properties. This allows the facile detection of novel compounds with potential medical applications. Remarkably, these biosensors are able to recognize the pharmacological properties that a particular hormone analogue exhibits in higher animals. Finally, the catalytic activity of our reporter enzyme was found to behave as a molecular sensor of the conformations of the hormone receptor, thus easily providing structural information that is very laborious to acquire with crystallographic methods.