Coral Fluorescent Proteins Figure 1. Excitation (solid lines) and emission (dashed lines) spectra of the newly cloned GFP-like proteins. Horizontal axis: wavelength in nanometers, vertical axis: relative fluorescence amplitude. doi:10.1371/journal.pone.0002680.g001 chromophore isomerization in chromoproteins, which converts them into far-red fluorescent proteins with the emission maximum above 600 nm. Although the quantum yield in such mutants is typically low, they show sufficient brightness to be considered useful biotechnology markers since they retain a high ‘‘chromoprotein- like’’ molar extinction. Another useful feature of the chromoproteins is the ‘‘kindling’’ behavior [19,28], which makes them prospective photoactivatable markers. All but one chromoprotein from the order Scleractinia identified previously, as well as in this study, are very similar in sequence and fall into the same phylogenetic group. The only significantly different chromoprotein is the novel pink spisCP from Stylophora pistilata. Its absorption maximum is at 560 nm (Fig. 1), which represents a blue shift by at least 14 nanometers in comparison to other known coral chromoproteins (hence pink rather than purple appearance). It should be noted, however, that some of the chromoproteins that arose independently in sea anemones (order Actiniaria) possess similarly blue-shifted absorption: for example, chromoproteins asCP562 [29] and cgCP [30] possess absorption maxima at 562 and 571 nm, respectively. Chromo-red. In addition to the above color classes proposed by Labas et al (2002), in this study we identified a protein eforRFP/CP from Echinopora forskaliana possessing rather unusual spectroscopic characteristics. This protein has a molar extinction coefficient exceeding 100,000 M21, which is more typical of chromoproteins than of red fluorescent proteins; however, it also shows the considerable (0.16) quantum yield of red fluorescence. The fluorescence peaks at 609 nm (Fig. 1), which is never seen in wild-type coral red fluorescent proteins (all of which emit below 600 nm), but is rather typical of fluorescent mutants of chromoroteins along with the relatively low quantum yield [19,30]. Its alkaline and acid denaturation behavior suggests the presence of a DsRed-like chromophore (not shown). Since the spectroscopic characteristics of this protein most closely resemble an artificially generated fluorescent mutant of a chromoprotein PLoS ONE | www.plosone.org 4 July 2008 | Volume 3 | Issue 7 | e2680