In work published in the June 2011 issue of Experimental Biology and Medicine, Kolossov, Spring and their co-investigators - a multidisciplinary team within the Institute for Genomic Biology at the University of Illinois - have transferred the concept of redox-sensitive Green Fluorescent Proteins (GFPs) to a quantitative Frster resonance energy transfer (FRET) imaging platform. For the FRET-based sensors, a change in redox induces a conformational change in a redox-sensitive switch that links two fluorescent proteins (the donor and acceptor), changing their distance, which in turn causes a detectable change in FRET efficiency. In its oxidized state the wavelength spectrum of the sensor's fluorescence emission is red-shifted (due to increased acceptor fluorescence), independent of variations in the local sensor concentration or in the intensity of the excitation light. As explained by Robert Clegg, a pioneer in the development of novel applications of optical microscopy in the biological sciences and key collaborator on the study, "FRET-based sensors circumvent the complications associated with imaging methods based on fluorescence intensity, since the increase in the FRET acceptor molecule's fluorescence can only take place if there is a change in the efficiency of energy transfer. This specific and discriminatory feature of FRET is one of the driving motives behind our development of a FRET-based assay rather than relying only on changes in the fluorescent intensity of a single component."
The current publication builds on the authors' previous work, where they reported a series of first-generation redox-sensitive linkers flanked by FRET donor and acceptor GFP-variants. As summarized by co-author Vladimir Kolossov, "The major advance in the current study is an improved dynamic range of the spectroscopic signal; in other words, a greater difference between fully reduced and oxidized states. Increasing the dynamic range leads to better discrimin
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