Fluorescence lifetime imaging of Myoglobin formation due to nitric oxide stress.

Myoglobin is a protein that is expressed quite unevenly among different cell types. Nevertheless, it has been widely acknowledged that the Fe state of myoglobin, myoglobin (Mb) has a broad functional role in metabolism, oxidative/nitrative regulation and gene networks. Accordingly, real-time monitoring of oxygenated, deoxygenated and Mb proportions- or, more broadly, of the mechanisms by which Mb is formed, presents a promising line of research. We had previously introduced a Förster resonance energy transfer (FRET) method to read out the deoxygenation/oxygenation states of myoglobin, by creating the targetable oxygen (O) sensor Myoglobin-mCherry. In this sensor, changes in myoglobin absorbance features that occur with lost O occupancy -or upon Mb production- control the FRET rate from the fluorescent protein to myoglobin. When O is bound, mCherry fluorescence is only slightly quenched, but if either O is released or is produced, FRET will increase- and this rate competing with emission reduces both emission yield and lifetime. Nitric oxide (NO) is an important signal (but also a toxic molecule) that can oxidize myoglobin to Mb with absorbance increases in the red visible range. mCherry thus senses both and deoxygenated myoglobin, which cannot be easily separated at hypoxia. In order to dissect this, we treat cells with NO and investigate how the Myoglobin-mCherry lifetime is affected by generating Mb. More discriminatory power is then achieved when the fluorescent protein EYFP is added to Myoglobin-mCherry, creating a sandwich probe whose lifetime can selectively respond to Mb while being indifferent to O occupancy.