Rhodospirillum rubrum responds to the presence of carbon monoxide (CO) in its environment by expressing a set of genes whose products oxidize CO to CO2. CooA is the protein that senses the presence of both reducing conditions and CO and responds by activating CO-dependent transcription. CooA is a homodimeric, heme-containing protein that is a member of the CRP/FNR family of activators. CooA exists in an equilibrium between active and inactive forms, and only the binding of CO to the heme of CooA shifts that equilibrium toward the active form. It is our working hypothesis that CO binding causes this activation by reorienting the heme, which in turn causes a repositioning of the adjacent long alpha helices at the dimmer interface. This helix repositioning transmits the signal of CO binding through the protein to stabilize the active conformation of the DNA-binding domains. The present proposal will test many aspects of this working model. This analysis will involve a variety of mutational analyses, both targeted changes informed by past results, as well as randomized mutageneses coupled with powerful genetic screens. CooA variants with interesting properties will purified and characterized functionally and spectrally in vitro to determine the molecular basis for their properties. Through this we will better define (i) the molecular basis for the specificity of CooA activation by CO; (ii) the nature of the signal transduction mechanism within CooA; (iii) the biochemical basis for the equilibrium between the active and inactive states; (iv) the driving force behind the conformational change that activates the protein; and (v) the basis for the interactions of CooA with RNA polymerase and with specific DNA sequences.The dramatic increase in our understanding of CooA in the recent grant period has made it an excellent model system for understanding the shared behavior within the CRP family, some of which are involved in regulation of virulence in other organisms. As a readily assessed transcriptional regulator, CooA presents distinct technical advantages compared with more medically relevant hemeproteins such as hemoglobin, P-450 and guanylyl cyclase, and its analysis will provide insights into the functional importance of hemeligands in other proteins as well. Finally, given the increasing evidence for CO as a messenger molecule in mammalian systems, CooA serves as a particularly tractable model system for understanding CO sensing in a biological system.
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