Color transitions in coral's fluorescent proteins by site-directed mutagenesis
© Gurskaya et al; licensee BioMed Central Ltd. 2001
Received: 4 May 2001
Accepted: 10 July 2001
Published: 10 July 2001
Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (~ 485 nm), green (~ 505 nm), yellow (~ 540 nm), and red (>580 nm) emitters.
Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence.
Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals.
Since the introduction of Green Fluorescent Protein (GFP) into biotechnology many attempts have been made to change its fluorescence color by means of mutagenesis. Finally, only two positions on protein sequence were demonstrated to considerably influence emission maximum [1,2,3]. First, a replacement of fluorophore's Tyr-66 with any aromatic residue (Trp, Phe, or His) results in a strong blue shift of emission up to λmax = 442 nm [4, 5]. Second, a substitution S203Y,H leads to a red shift of fluorescence up to λmax = 529 nm . These blue and yellow mutants (called BFP and YFP respectively) proved to be extremely handy for multicolor labeling and FRET-based applications.
Recently we have isolated several GFP-like proteins that determine fluorescent or non-fluorescent body color in corals Anthozoa [7,8,9]. Absorption-emission maxima of these proteins are distributed loosely along the wavelength axis. These proteins fall into four groups: cyan (~ 485 nm), green (~ 505 nm), yellow (~ 540 nm), and red (>580 nm) emitters. Obviously, understanding the relation between protein structure and its fluorescent properties is of great scientific and practical importance. However, in spite of the abundance of sequences available, it is difficult to determine amino acid positions that are responsible for a particular type of fluorescence.
Results and discussion
Blue shift for cyan FP amFP486
Comparison of the spectral properties of zFP506, amFP486, and their mutants at position 167.
Excitation maximum, nm
Excitation spectral width, nm
Emission maximum, nm
Emission spectral width, nm
In one particular case, two closely related FPs of different colors coexist in a single organism, Zoanthus sp. . One of them, zFP506, displayed green fluorescence and the other, zFP538, - yellow fluorescence. Having 87% identity to each other, these proteins are an excellent model for hunting amino acids required for green-yellow transitions. Most of 31 amino acids that distinguish zFP506 from zFP538 are external and only 8 of these residues are buried, assuming that structures of Anthozoa FPs are similar to the β-can fold of GFP [6, 11]. Four out of eight buried amino acid disparities are grouped in proximity of chromophore-forming residues at positions 66 and 67 (Fig. 1). It is reasonable to suppose that some of these residues are the cause of the color difference.
Accidentally, our other mutagenesis project on randomization of position 65 in several FPs (this work will be published elsewhere) revealed Lys-65 in zFP538 as a key residue responsible for the yellow fluorescence. Among seven randomly tested amino acid substitutions at position 65 (Leu, Ile, Met, His, Gln, Asn, or Lys) only the wild type variant Lys-65 emitted yellow light. Other mutants displayed green fluorescence or lacked fluorescent properties. We characterized in detail the brightest green-emitting mutant containing a single amino acid substitution K65M. In terms of brightness, shape and maxima of excitation-emission spectra this mutant protein was estimated to be practically undistinguishable from zFP506 (Fig. 2C). The finding that Lys-65 determines the yellow color of fluorescence was unexpected as Lys in this position is not unique for zFP538: one cyan emitter, amFP486, hosts Lys-65 (Fig. 1). Thus, one can conclude that only a simultaneous action of Lys-65 together with some other residue(s) results in a red shift of fluorescence.
Then, we paid attention to Asp-68 that is unique for zFP538. A substitution D68N resulted in dual-peak (green and yellow) fluorescence (Fig. 2D). So, Asp-68 in zFP538 is important but not vital for yellow fluorescence.
Finally, we attempted to convert zFP506 into a yellow emitter. Unexpectedly, two of the replacements discussed above were not sufficient for this transition. A mutant protein N65K/N68D possessed green fluorescence similar to the wild type protein's (not shown). Only a triple mutant of zFP506 carrying substitutions A63G/N65K/N68D displayed emission maximum in yellow region at 537 nm (Fig. 2E). In comparison with the natural yellow emitter zFP538 this mutant protein had slightly modified excitation spectrum: the minor peak at 494 nm became more pronounced.
Summing up, we performed the following transitions of fluorescent colors for zFP506 and zFP538 proteins: (i) yellow to green, (ii) yellow to dual color (green and yellow), and (iii) green to yellow. Switch-like color transitions as well as dual-color fluorescence may indicate that the green and yellow fluorescence represent chemically distinct fluorophores. Our hypothesis is that a positive charge of Lys-65 and a negative charge of Asp-68 are involved in catalysis at yellow fluorophore formation, while Gly-63 provides the required steric freedom.
Recently, the structure of drFP583 "red" chromophore was determined . Crystallographic studies of drFP583 supported this model of chromophore structure [13, 14]. In comparison with GFP-type green chromophore, red one was found to contain a more extended π-system due to the presence of a double bond between the α-carbon and the nitrogen of Gln-65. Thus, the red chromophore matures through a green GFP-like intermediate by means of additional autocatalytic step of dehydrogenation. Mutagenetic studies of red FPs, drFP583 and asFP595, revealed many point mutations that transform them into dual-color (green and red) or even pure green emitters [9, 15, 16]. So, red fluorescent color can be very easily converted into green by a disruption of the chromophore's environment required for the last autocatalytic step. Here we attempted to create such "red" environment in a cyan FP. Serine residues at positions 68 and 112 were found to be essential for red chromophore formation, but are not sufficient to completely convert cyan FP into red: only a small portion of the mutant protein becomes red emitting. Obviously, an elaborate set of amino acids should surround the chromophore to ensure the maturation of the "red" structure. According to the crystal structure of drFP583, Ser-68 is located in the closest proximity to the double bond between α-carbon and nitrogen of Gln-65 specific for the red fluorophore [13, 14]. One is tempted to suppose that Ser-68 plays some catalytic role in formation of this double bond. It is more difficult to explain significance of Ser-112 because this residue is situated rather far from chromophore. However, Ser-112 is bound through water molecules with oxygen atoms of both Gly-67 and the imidazolidinone ring. These hydrogen bonds could be important for proper chromophore positioning or could be a pathway for proton transfer.
Based on the results reported here we believe it is possible to predict the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals. The most characteristic point is position 68. At this position cyan and green FPs should contain Asn or a hydrophobic residue; yellow FPs should contain Asp or possibly Glu (negatively charged residues); red FPs and asFP595-like chromoproteins should contain Ser. Then, position 167 probably allows to discriminate cyan and green emitters: bulky aliphatic residues (e.g. Met, He, Leu) are characteristic for green FPs and polar or small residues (e.g. His, Ala) are specific for cyan FPs. However, a more extended list of FPs of different colors is required to prove or contradict our conclusions.
Materials and Methods
Site-directed mutagenesis was performed by PCR using the overlap extension method, with primers containing appropriate target substitutions . All mutants were cloned into pQE30 vector (Qiagen) using BamHI and SalI restriction sites. Recombinant proteins contained 6xHis tag on N-terminus. E. coli clones were grown at 37°C in 50 ml to an optical density of (OD) 0.6. At that point, the expression of recombinant FP was induced with 0.2 mM IPTG. The cultures were then incubated overnight. The following day, cells were harvested by centrifugation, resuspended in buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl), and disrupted by sonication. Fluorescent proteins were purified from the soluble fraction using TALON Metal Affinity Resin (CLONTECH). Proteins were at least 95% pure according to SDS-PAGE. Perkin-Elmer LS50B spectrometer was used for spectral measurements of the purified fluorescent proteins diluted in buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl) up to about 50 μg/ml. The excitation and emission slits were set at 5 nm. The spectra were corrected for photomultiplier response and monochromator transmittance.
We are grateful to M. V. Matz for fruitful discussion, M. E. Bulina for the help in the manuscript preparation, and S. J. Remington and R. Y. Tsien for communication of data prior to publication. This work was supported by CLONTECH Laboratories Inc. and the Russian Foundation for Fundamental Research (grant 01-04-49037).
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