The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.

Fluorescent protein (FP) variants that can be reversibly converted between fluorescent and nonfluorescent states have proven to be a catalyst for innovation in the field of fluorescence microscopy. However, the structural basis of the process remains poorly understood. High-resolution structures of a FP derived from Clavularia in both the fluorescent and the light-induced nonfluorescent states reveal that the rapid and complete loss of fluorescence observed upon illumination with 450-nm light results from cistrans isomerization of the chromophore. The photoinduced change in configuration from the well ordered cis isomer to the highly nonplanar and disordered trans isomer is accompanied by a dramatic rearrangement of internal side chains. Taken together, the structures provide an explanation for the loss of fluorescence upon illumination, the slow light-independent recovery, and the rapid light-induced recovery of fluorescence. The fundamental mechanism appears to be common to all of the photoactivatable and reversibly photoswitchable FPs reported to date.crystallography ͉ fluorescence ͉ photoswitching ͉ protein structure G reen fluorescent protein (GFP) from the jellyfish Aequorea victoria (1) and more recently, cyan, yellow, and red fluorescent proteins (FPs), isolated from coral reef organisms (2), have become standard research tools in cellular biology. Structural studies have revealed a universally conserved fold for FPs: an 11-stranded ␤-barrel with a central and axial ␣-helix (3, 4). A sequence of three amino acid residues on this central ␣-helix (Ser-65-Tyr-66-Gly-67 in Aequorea GFP) undergoes a series of autocatalytic posttranslational modifications to form the chromophore. Despite overall structural similarity across FPs from a variety of species and a variety of hues, members of the GFP family are quite diverse with respect to their photophysical properties. For example, properties such as emission color, extinction coefficient, quantum yield, and fluorescence lifetime can vary dramatically among FP variants. These properties are determined by both the influence of the protein matrix on the chromophore environment and the particular covalent chemical structure of the chromophore (5).Another photophysical property common to all FPs is wavelength-dependent change in their f luorescence emission properties upon illumination. In most cases, these changes appear to result from irreversible covalent modification of the chromophore structure or its local protein environment. Examples of such permanent changes include light-induced transition from a f luorescent to a nonf luorescent state (photobleaching), transition from a nonf luorescent to a f luorescent state (photoactivation), or a change in excitation or emission wavelength maxima (photoconversion). Photobleaching, photoactivation, and photoconversion are well documented and can be advantageous for f luorescence imaging applications such as in vivo tracking of fusion protein movements (6 -9). A less well documented process, occasionally described as ''re...

Fluorescent proteins isolated from coral reef organisms can be roughly grouped into four color classes by emission, cyan, green, yellow, and red. To gain insight into the structural basis for cyan emission, the crystal structure of amFP486 ( em max ‫؍‬ 486 nm) was determined by molecular replacement, and the model was refined at 1.65-Å resolution. The electron density map reveals a chromophore formed from the tripeptide sequence -K-Y-G-that is indistinguishable from that of GFP ( em max ‫؍‬ 509 nm). However, the chromophore environment closely parallels those of the yellowand red-shifted homologs zFP538, DsRed, and eqFP611. Mutagenesis was performed for Glu-150, Ala-165, His-199, and Glu-217, which are immediately adjacent to the chromophore. His-199 and Ala-165 are key side chains responsible for the blue shift, presumably by localizing chromophore charge density on the phenolate moiety. Furthermore, in the H199T mutant the fluorescence quantum yield is reduced by a factor of Ϸ110. The crystal structures of H199T ( em max ‫؍‬ 515 nm) and E150Q ( em max ‫؍‬ 506 nm) were determined. Remarkably, the H199T structure reveals that the stacking interaction of His-199 with the chromophore also controls the fluorescence efficiency, because the chromophore is statistically distributed in a 1:1 ratio between cis (fluorescent) and trans (nonfluorescent) conformations.autocatalysis ͉ fluorescent proteins ͉ protein crystallography T he discovery in Anthozoa of an entire family of fluorescent proteins (FPs) and nonfluorescent chromoproteins distantly related to the green FP from Aequorea victoria (GFP) has provided new tools that can serve as alternatives to or complement the existing uses of GFP (1-5). Based on absorbance and emission spectra, the Anthozoa proteins can be roughly grouped into five classes: cyan, green, yellow, red, and nonfluorescent chromoproteins. The amino acid sequences are closely related, and in all known cases, a tripeptide -X-Y-G-, where X is highly variable, forms the precursor to the chromophore. The large spectral diversity appears to arise from two causes, the most important being differences in the extent of bond conjugation arising from variations in the chemical structure of the chromophore. For the yellow-and red-emitting FPs, the spectral diversity results from oxidation of the polypeptide backbone to form an acylimine linkage or variations thereof [e.g., DsRed (6-8); eqFP611 (9), zFP538 (10), and the ''kindling FP'' KFP (11, 12); for chromophore structures, see Fig. 6, which is published as supporting information on the PNAS web site]. However, effects arising from the local environment of the chromophore can have a significant influence on absorption and emission maxima. Variations of up to Ϸ20 nm in either direction have been achieved by means of single-site substitutions in GFP (13,14) and in DsRed (15).Three cyan-emitting Anthozoa FPs (CFPs) [amFP486, dsFP483. and cFP484 (1)] have been characterized in some detail. Although the biological role of these proteins is unknown, due to widesp...

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mKeima is an unusual monomeric red fluorescent protein (λemmax ~620 nm) that is maximally excited in the blue (λexmax ~440 nm). The large Stokes shift suggests that the chromophore is normally protonated. A 1.63 Å resolution structure of mKeima reveals the chromophore to be imbedded in a novel hydrogen bond network, different than in GFP, which could support proton transfer from the chromophore hydroxyl, via Ser142, to Asp157. At low temperatures the emission contains a green component (λemmax ~535 nm), enhanced by deuterium substitution, presumably resulting from reduced proton transfer efficiency. Ultrafast pump/probe studies reveal a rising component in the 610 nm emission with lifetime ~4 ps, characterizing the rate of proton transfer. Mutation of Asp157 to neutral Asn changes the chromophore resting charge state to anionic (λexmax ~565 nm, λemmax ~620 nm). Thus, excited state proton transfer (ESPT) explains the large Stokes shift. This work unambiguously characterizes green emission from the protonated acylimine chromophore of red fluorescent proteins.