[11,17]. In addition, there is a clear difference in the absorption spectrum of these types of red proteins under denaturing conditions. In 1M NaOH a DsRed chromophore is hydrolyzed resulting in a green-type chromophore structure with the characteristic absorption maximum at 445 nm [15]. In contrast, a Kaede-type chromophore in 1M NaOH absorbs with the maximum at 499 nm [10]. Kaede-type red proteins show a peculiar photo-induced color conversion: their final chromophore maturation stage that transforms the green-emitting GFP-like structure into the red chromophore requires violet light [16]. The photoconversion feature of Kaede-like red FPs made it possible to evaluate the half-life of these proteins in vivo, which turned out to be extremely long-on the order of 20 days [18]. Non-fluorescent purple-blue proteins (so-called chromoproteins or pocilloporins), which are characterized by high molar extinction coefficient but virtually no fluorescence [7,19], possess yet another chromophore type, which is the isomerized version of the DsRed-like red chromophore [20]. There are three more derivatives of the DsRed-like structure, each observed once among FPs. The first one is the fragmented DsRed chromophore of the kindling fluorescent protein (KFP) from the sea anemone Anemonia sulcata [21], which was originally described as a chromoprotein [19]. Another one is a three-ring structure [22] found in the natural yellow fluorescent protein from Zoanthus sp. [23]. The third one is found in a mutant variant of DsRed called mOrange [24]. It is still largely unclear how changes in the FP’s amino acid sequence lead to such dramatic variations. We have earlier reported the detailed analysis of remote homology relationships within the GFP superfamily [13,25]. The aim of the present study was to systematically characterize fluorescent and/or colored GFP-like proteins found in reef- building corals (phylum Cnidaria, class Anthozoa, order Scler- actinia), which represent the largest known repository of spectro- scopic diversity of the GFP-like proteins. We describe 40 novel proteins that, along with the previously known ones, represent sampling from all six suborders of Scleractinia and cover 14 out of 21 families of corals. Our study provides an extensive knowledge base for biotechnological, evolutionary, and ecological studies utilizing GFP-like proteins as a subject or as a model. Results General characteristics of coral FPs We used six sets of degenerate primers targeting the whole previously known diversity of Anthozoan FPs to isolate the coral FPs described here. Every coral species was subject to PCR trials with all degenerate primer combinations, resulting in identification of up to four distinct FPs from a single species. All but one of the newly cloned proteins fall into one of the four previously suggested color classes: fluorescent cyan, green, and red proteins, and non- fluorescent chromoproteins [5,9]. A novel color type was represented by the protein from Echinopora forskaliana that exhibited the spectral phenotype intermediate between chromoproteins and DsRed-type red fluorescent proteins (hence its identifier is eforCP/ RFP). Table 1 summarizes the spectral characteristics of the proteins cloned in this study, while Figure 1 shows all the excitation and emission spectra. According to the conservative method of semi-native gel electrophoresis [26], all the newly cloned FPs are tetramers or higher-order oligomers. Colors Cyan. Although cyan proteins possess the same chromophore as greens [14], their evolution in corals by means of positive natural selection [12] warrants their recognition as a separate color class Coral Fluorescent Proteins since it indicates that cyan fluorescence must have a dedicated, although yet unclear, role in corals’ physiology. Cyan proteins typically have an emission peak between 485–495 nm, although more blue-sifted variants can occasionally be found, down to 477 nm [27] . The considerable variation in exact position of the maxima is in a large part due to the poorly defined peaks in the spectral curves. Cyan proteins have notably wider excitation and emission curves than greens: the width of the curves at half-height is about 55 nm for cyans compared to about 35 nm for greens. Cyan proteins typically show the lowest molar extinction coefficient of all the colors (Table 1). Two cyan proteins reported here (psamCFP and mmilCFP) exhibit dramatically blue-shifted excitation maximum (404 nm), suggestive of a predominantly neutral ground-state chromophore, which, as we show below, is due to the presence of glutamic acid in position 167 (numeration according to GFP from Aequorea victoria). This spectral modification was previously unknown in cyan fluorescent proteins, either wild-type or artificially generated mutant variants. Green. Green fluorescent color is the most common in corals and is the most conspicuous of all the fluorescent colors in situ [8]. We discriminate green proteins from the cyans by position of emission maximum (.500 nm), which usually correlates with the narrow half-width of the excitation and emission curves (see above). Occasionally there are borderline cases such as aacuGFP1, which has a narrow ‘‘green-like’’ emission peak at 502 nm but a rather ‘‘cyan-like’’ blue-shifted and wide excitation peak (Fig. 1). The position of excitation maxima in the newly cloned green proteins (around 478–512 nm) indicates the predominance of an anionic ground chromophore state, although a group of closely related proteins from Acropora species (aacuGFP1, aacuGFP2, amilGFP and anobGFP) display a minor, but noticeable absorption peak at about 395 nm corresponding to the neutral chromophore state. Yellow. There are two wild-type yellow fluorescent proteins with emission maxima between 525 and 570 nm known at the moment: zoanYFP from a Zoanthidea representative (emission max 538 nm) and a hydromedusan protein phiYFP (emission max 535 nm). Despite the significant extent of our survey, not a single protein of this color has been cloned from corals, although yellow fluorescence with emission maximum exceeding 530 nm has been occasionally observed [17]. zoanYFP and phiYFP represent two different solutions to achieve yellow fluorescence. While phiYFP contains a GFP-like chromophore in a modified environment, zoanYFP possesses a unique three-ring chromophore structure that seems to be a result of deviation from the DsRed-type chromophore synthesis pathway. Interestingly, as it will be discussed below, such an explanation is corroborated by the phylogenetic position of zoanYFP. Red. Corals possess either DsRed-type or Kaede-type red fluorescent proteins. We find Kaede-type proteins mostly associated with scleractinian corals of suborder Faviina. In addition, Kaede- type proteins are found in at least one representative of the order Corallimorpharia (carribean mushroom anemone Ricordea florida) and at least one representative of the family Nephtiidae of the order Alcyonaria, Dendronephtya sp. Red fluorescent proteins from all other organisms studied thus far, including other suborders of reef-building corals (Scleractinia), all sea anemones (Actiniaria) and two more Corallimorpharia representatives, possess the DsRed-like chromophore. Purple-blue. The non-fluorescent chromoproteins (also called pocilloporins [7]) are characterized by intense absorption with a molar extinction coefficient commonly exceeding 100,000 and virtually no fluorescence. The chromophore in the chromoproteins is an isomerized non-coplanar version of the DsRed-like chromophore [20]. A mutation was previously identified that results in the PLoS ONE | www.plosone.org 2 July 2008 | Volume 3 | Issue 7 | e2680