| Signal transduction networks mediate cellular responses to environmental stimuli. These signals may be chemical or physical in nature and include light, chemical ligands and electric field. My thesis addresses how signals are transmitted at the molecular level by a family of sensor proteins containing Per-ARNT-Sim (PAS) sensor domains. PAS sensor domains comprise a superfamily of more than 20,000 members found in all kingdoms of life. Interestingly, despite having a conserved fold, PAS domains recognize and respond to a range of signals including light, oxygen and redox potential and couple these signal inputs to output behaviors either directly or indirectly, through an associated effector domain. The widespread and modular nature of PAS sensor proteins, and their ability to sense many different types of environmental stimuli and linkage to a wide range of effector (output) domains, make them an ideal system for studying the rules governing signal transduction in cells and remodeling input/output (I/O) signaling processes of cells.;PAS sensor proteins represent a toolkit of "plug-and-play" parts (i.e., modules or domains) that can be used to remodel cellular behavior. For example, the PAS sensor protein FixL from Bradyrhizobium japonicum (BjFixL), which mediates nitrogen fixation in root nodules of plants, is comprised of an oxygen-sensitive heme PAS domain covalently linked to a second N-terminal PAS domain and a C-terminal histidine kinase effector domain. On the other hand, a photosensory PAS domain from Bacillus subtilis YtvA (BsYtvA-LOV), linked N-terminally to a STAS domain, binds FMN and senses light. By replacing the heme PAS sensor domain of BjFixL with BsYtvA-LOV, the signaling specificity of BjFixL can be switched from oxygen to light (Moglich et al., 2009a).;In this thesis, I address multiple aspects of PAS sensor protein signaling including: (i) the role of quaternary structure in the signaling mechanisms of PAS sensor proteins, (ii) engineering novel two-input PAS sensor proteins, (iii) the development of a light-regulated transcription system in Escherichia coli on the basis of an engineered, light-sensitive, PAS sensor kinase, and (iv) the identification of an evolutionarily-conserved network of residues in PAS domains that may form the basis of structural and functional similarities among members of the PAS domain superfamily.;Understanding how PAS sensor proteins signal will inform the underlying signaling networks that govern cell morphology, seed germination, phototaxis, and many other physiological behaviors. Moreover, these rules may be used to engineer novel protein sensors that, when integrated into a cellular system, re-wire the response of a cell to an environmental signal. |
