Although high levels of carbon monoxide are known to be toxic to most organisms it has been found that local, low levels of carbon monoxide (CO) and nitric oxide are implicated in a wealth of cellular signaling pathways and mechanisms.  The exact role of carbon monoxide in these pathways remains opaque however, due to the difficulty in detecting carbon monoxide concentration fluctuations in living cells.  Certain heme binding proteins, such as the CO sensing protein CooA from the organism R. rubrum, bind CO very selectively and then undergo a large conformational change.  Recently, researchers inserted a circularly permuted green fluorescent protein (GFP) into the hinge region of the CooA protein, which is the region that undergoes the conformational change following CO binding.  This construct was shown to exhibit a shift in fluorescence signal following CO binding in live cells.  In this work, we constructed a CO sensor by inserting a solvatochromic fluorophore into the hinge region of the CooA protein.  The solvatochromic fluorophore was inserted into the CooA protein using site-specific in vivo unnatural amino acid incorporation, in which a stop codon was inserted into a site that corresponded to an area in the hinge region that changed polarity following CO binding.  This sensor has an advantage over the previously developed GFP version in that the fluorophore is a small organic moiety, instead of a large protein, thereby reducing the potential steric interferences and mechanistic perturbations that a large chimera may introduce into a living system.

Developing a CO sensor via unnatural amino acid incorporation into heme proteins

248th National Meeting (2014)

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