The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor

Abstract

We have crystallized and subsequently determined to 2.0-Å resolution the crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. The structure of the protomer, which adopts a β-can topology, is similar to that of the related monomeric green fluorescent protein (GFP). The quaternary structure of eqFP611, a tetramer exhibiting 222 symmetry, is similar to that observed for the more closely related red fluorescent protein DsRed and the chromoprotein Rtms5. The unique chromophore sequence (Met63-Tyr64-Gly65) of eqFP611, adopts a coplanar and trans conformation within the interior of the β-can fold. Accordingly, the eqFP611 chromophore adopts a significantly different conformation in comparison to the chromophore conformation observed in GFP, DsRed, and Rtms5. The coplanar chromophore conformation and its immediate environment provide a structural basis for the far red, highly fluorescent nature of eqFP611. The eqFP611 structure extends our knowledge on the range of conformations a chromophore can adopt within closely related members of the green fluorescent protein family. We have crystallized and subsequently determined to 2.0-Å resolution the crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. The structure of the protomer, which adopts a β-can topology, is similar to that of the related monomeric green fluorescent protein (GFP). The quaternary structure of eqFP611, a tetramer exhibiting 222 symmetry, is similar to that observed for the more closely related red fluorescent protein DsRed and the chromoprotein Rtms5. The unique chromophore sequence (Met63-Tyr64-Gly65) of eqFP611, adopts a coplanar and trans conformation within the interior of the β-can fold. Accordingly, the eqFP611 chromophore adopts a significantly different conformation in comparison to the chromophore conformation observed in GFP, DsRed, and Rtms5. The coplanar chromophore conformation and its immediate environment provide a structural basis for the far red, highly fluorescent nature of eqFP611. The eqFP611 structure extends our knowledge on the range of conformations a chromophore can adopt within closely related members of the green fluorescent protein family. The green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; r.m.s., root mean-squared. from Aequorea victoria has generated widespread interest as a biotechnological tool, acting as a visual reporter for events in living cells. With a view to increasing potential uses for GFP, considerable effort has been invested to evolve new variants of GFP (1Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4945) Google Scholar). For example, GFP variants and GFP homologs have been reported that together cover almost the entire visible range of emission wavelengths (420–630 nm) (1Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4945) Google Scholar, 2Matz M.V. Fradkov A.F. Labas Y.A. Savitsky A.P. Zaraisky A.G. Markelov M.L. Lukyanov S.A. Nat. Biotechnol. 1999; 17: 969-973Crossref PubMed Scopus (1528) Google Scholar, 3Gurskaya N.G. Fradkov A.F. Terskikh A. Matz M.V. Labas Y.A. Martynov V.I. Yanushevich Y.G. Lukyanov K.A. Lukyanov S.A. FEBS Lett. 2001; 507: 16-20Crossref PubMed Scopus (234) Google Scholar) while other variants have been optimized or are suitable as biosensors for pH (4Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1972) Google Scholar), redox potential (5Ostergaard H. Henriksen A. Hansen F.G. Winther J.R. EMBO J. 2001; 20: 5853-5862Crossref PubMed Scopus (261) Google Scholar), or Ca2+ ions (6Miyawaki A. Griesbeck O. Heim R. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2135-2140Crossref PubMed Scopus (727) Google Scholar). In addition to using protein engineering approaches, GFP-like proteins have been isolated from alternative natural sources, exhibiting spectroscopic properties that could be of value as reporters. For example, the range of colors available has been extended with the discovery of naturally occurring red-emitting homologues such as DsRed isolated from the corallimorphian Discosoma sp (2Matz M.V. Fradkov A.F. Labas Y.A. Savitsky A.P. Zaraisky A.G. Markelov M.L. Lukyanov S.A. Nat. Biotechnol. 1999; 17: 969-973Crossref PubMed Scopus (1528) Google Scholar) and eqFP611 from the sea anemone Entacmaea quadricolor (8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar). Applications using DsRed as a fluorescent marker have been limited however, because of a number of undesirable properties including its oligomeric nature and slow chromophore maturation. Nevertheless, extensive random mutagenesis of DsRed has produced variants with much improved properties (9Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (2008) Google Scholar, 10Bevis B.J. Glick B.S. Nat. Biotechnol. 2002; 20: 83-87Crossref PubMed Scopus (499) Google Scholar). Other fluorescent proteins have been isolated such as the Kaede protein with striking photoresponsive behavior (11Ando R. Hama H. Yamamoto-Hino M. Mizuno H. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2651-2656Crossref Scopus (832) Google Scholar). In addition, a range of weakly and non-fluorescent chromoproteins, including the pocilloporin Rtms5 from the reef building coral Montipora efflorescens (12Prescott M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure. 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and the GFP homologue asFP595 from Anemonia sulcata (13Lukyanov K.A. Fradkov A.F. Gurskaya N.G. Matz M.V. Labas Y.A. Savitsky A.P. Markelov M.L. Zaraisky A.G. Zhao X Fang Y. Tan W. Lukyanov S.A. J. Biol. Chem. 2000; 275: 25879-25882Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) have been described. Some of these proteins have important biological roles (14Dove S.G. Hoegh-Guldberg O. Ranganathan S. Coral Reefs. 2001; 19: 197-204Crossref Scopus (209) Google Scholar). For example, the vividly colored yet often poorly fluorescent pocilloporins possess multiple photoprotective functions, helping to protect the photosystems of their resident microalgae from high-amplitude light fluctuations that can lead to severe photoinhibition (15Salih A. Larkum A. Cox G. Kuhl M. Hoegh-Guldberg O. Nature. 2000; 408: 850-853Crossref PubMed Scopus (483) Google Scholar, 16Jones R. Hoegh-Guldberg O. Plant Cell Environ. 2001; 24: 89-100Crossref Scopus (165) Google Scholar). Aside from biotechnology-based considerations, of fundamental interest is the structural basis for the similarities and differences of the spectral properties these fluorescent proteins and chromoproteins exhibit. One common feature is the presence of an extended, conjugated π-system comprising a cyclic tripeptide chromophore (Ser-Tyr-Gly in GFP; Gln-Tyr-Gly in DsRed and Rtms5) buried within the distinctive β-can topology. Differences in spectral properties arise from the unique environment in which the chromophore resides together with modifications to the π-resonance system. For example, in comparison to GFP, the red-shifted spectral properties of DsRed have been attributed to chemical modifications of the GFP-like chromophore that extend the coplanar π-resonance system (17Yarbrough D. Wachter R.M. Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 462-467Crossref PubMed Scopus (394) Google Scholar, 18Wall M.A. Socolich M. Ranganathan R. Nat. Struct. Biol. 2000; 7: 1133-1138Crossref PubMed Scopus (301) Google Scholar). However, despite possessing the same chromophore sequence as DsRed, Rtms5 adopts a markedly different conformation to that observed in DsRed, such that the phenoxy ring of the chromophore is in a trans and non-coplanar conformation (12Prescott M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure. 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The majority of fluorescent proteins with emission maxima significantly beyond 600 nm have been found to be weakly fluorescent. A notable exception is the highly fluorescent protein eqFP611 recently isolated from the sea anemone E. quadricolor that when excited, emits light maximally at 611 nm (8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar). In order to increase our understanding of the structural basis for protein fluorescence at the far red end of the color scale, we have determined the crystal structure of the highly fluorescent protein, eqFP611 to 2.0-Å resolution. The crystal structure of eqFP611 provides a detailed view of the chromophore and its environment, thereby providing a platform for the design of variants of eqFP611 with improved spectral and oligomeric properties, as well as clues for altering similar properties of other chromoproteins, such as Rtms5. Protein Expression and Purification—Single colonies of Escherichia coli (BL21-DE3) freshly transformed with a vector encoding eqFP611 with an N-terminal His6 tag (8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar) were inoculated into LB medium. Cells were incubated overnight with orbital shaking (200 rpm) at 28 °C. 2 ml of the overnight culture were transferred to 500 ml of 2YT medium and after 4 h incubation isopropyl-1-thio-β-d-galactopyranoside added to a final concentration of 0.16 mm. Incubation was continued overnight after which the cells were harvested. Recombinant protein was isolated and purified by Ni-nitrilotriacetic acid and gel filtration chromatography as described (19Beddoe T. Ling M. Dove S. Hoegh-Guldberg O. Devenish R.J. Prescott M. Rossjohn J. Acta Crystallogr. D. Biol. Crystallogr. 2003; 59: 597-599Crossref PubMed Scopus (20) Google Scholar). The majority of the protein eluted from a calibrated S200 size exclusion column with an apparent size (∼100 kDa.) that corresponded closely to the size predicted for the tetrameric form of eqFP611 (data not shown). Minor amounts of earlier eluting material indicated possible aggregation of the protein. These results confirm an earlier report that eqFP611 behaves as a tetramer when subjected to gel filtration chromatography at micromolar concentrations and under certain conditions of expression can form aggregates (8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar). Denaturing SDS-PAGE indicated a single polypeptide of M r 26,000. Column fractions corresponding to the tetramer were pooled and concentrated with a centrifugal ultrafiltration device (Millipore, MWCO 10,000) to 26 mg/ml. Crystallization—Crystals of eqFP611 were obtained at 20 °C using the hanging drop vapor diffusion method. Purified eqFP611 protein at 26 mg/ml was mixed with an equal volume of a reservoir solution containing 11.5% polyethylene glycol 8000, 0.1 m sodium acetate pH 4.3 and 0.2 m calcium acetate. Crystals formed after 1–3 days grew to maximal size between 7 and 14 days. The absorption and fluorescence excitation/emission spectra were determined for crystals redissolved at pH 8.0. Spectra were similar for eqFP611 solutions at pH 8.0 (purification buffer) and published values (data not shown, Ref. 8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar). Data Collection—The crystals were flash cooled during data collection using 15% PEG 400 as the cryoprotectant. A 2.0-Å data set was collected using a RU-3HBR x-ray source and an R-AXIS IV++ detector by inverse phi geometry. The data were merged and processed with the HKL software package (20Ottwinowski Z. Sawyer L. Issacs N. Bailey S. Data Collection and Processing. SERC Daresbury Laboratory, UK1993: 56-62Google Scholar). The crystals, with unit cell dimensions a = 78 b = 78 c = 328 Å belong to space group P6522. See Table I for a summary of statistics. V m calculations suggested there were two protomers per asymmetric unit. Inspection of the native Patterson map suggested that the two protomers had a common orientation within the asymmetric unit.Table IData collection and refinement statisticsData collection statisticsTemperature100KSpace GroupP6522Cell dimensions (Å) (a,b,c)77.9, 77.9, 327.5Resolution (Å)2.0Total no. of observations134722No. of unique observations37625Multiplicity3.6Data completeness (%)91.2 (79.7)No. data > 2σ176.9 (38.3)I/σ121.0 (2.9)R mergeaR merge = Σ |Ihkl - 〈Ihkl 〉|/ΣIhkl . (%)4.6 (23.0)Refinement statisticsNon-hydrogen atomsProtein3548Chromophore46Calcium2Acetate8Water285Resolution (Å)50-2.0R factorbR factor = Σhkl||Fo | - |Fe ||/Σhkl|Fo | for all data except for 4%, which was used for the R free calculation. (%)21.4R free (%)25.3r.m.s. deviations from idealityBond lengths (Å)0.007Bond angles (°)1.37Impropers (°)0.81Dihedrals (°)26.03Ramachandran plotMost favored91.0And allowed region (%)9.0B-factors (Å2)Average main chain37.0Average side chain39.5Average water R merge = Σ |Ihkl - 〈Ihkl 〉|/ΣIhkl R factor = Σhkl||Fo | - |Fe ||/Σhkl|Fo | for all data except for 4%, which was used for the R free calculation. in a new crystal structure of eqFP611 was determined using the with the in the A of the tetrameric DsRed structure Data was used as the with all sequence differences to and the chromophore A in the to the of two which well within the unit cell and formed a 222 tetramer the The of refinement was by the R free value of the with a a resolution to the The structure was using by the in J. M. R.J. T. Acta Crystallogr. D. Biol. Crystallogr. 1998; PubMed Scopus Google Scholar), with of building using the M. Acta A Scopus Google Scholar). were used from were from these refinement was and were to the data were in the were within to in at and had a The for the chromophore (Met63-Tyr64-Gly65) was and at the of was into the and for the chromophore were using and were to to the acid and The between the chromophore and to a trans is to that for the DsRed and Rtms5 The the to chromophore indicated that the region not possess the of a The the of the group was in improved of the into The the to from the value of The was set at the same observed in DsRed and Rtms5 and to an improved of the into The the chromophore indicated that the group was with the the of in the of of chromophore that the and to be were the refinement and building of eqFP611, a was observed in the map the of the the which was as a water However, refinement indicated that a water was to for the observed an acetate was of the The final which two protomers water 2 calcium and two acetate has an R factor of and an R free of for all between 20 and See Table I for summary of refinement and The and structure have been in the data The crystal structure of eqFP611 has been determined to 2.0-Å resolution Table of the two protomers within the asymmetric the is Å for all structural be to The eqFP611 which 222 symmetry, is generated structural be to Rtms5 and DsRed, homologues that in the red of the final the eqFP611 chromophore The eqFP611 has a similar to that described for GFP, DsRed, and a in which a that is from with the of the and the structural that the chromophore to the protein. with other members of the GFP are the structure of eqFP611 is similar to that of DsRed sequence an of and Rtms5 sequence an of while to GFP sequence an of The differences within the and and the the notable of which is a in eqFP611 which a with to Rtms5 and DsRed eqFP611 to be as by to DsRed and eqFP611 has a that at the A number of sequence and structural differences were observed to the chromophore which have a on the conformation and spectral properties of the The eqFP611 chromophore structure can be to that of DsRed and as a group The in the is observed to be and is with the of a between and at A similar was for the in DsRed and Rtms5 (12Prescott M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure. 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, D. Wachter R.M. Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 462-467Crossref PubMed Scopus (394) Google Scholar, 18Wall M.A. Socolich M. Ranganathan R. Nat. Struct. Biol. 2000; 7: 1133-1138Crossref PubMed Scopus (301) Google Scholar), is in extends the system of the eqFP611 chromophore and is with the red-shifted emission of the protein. is from the that the eqFP611 chromophore adopts a coplanar conformation 2 and The factor for the chromophore is similar to that of the side that there is limited of the which is with the number of the chromophore 2 and a of Table are notable differences in the chromophore conformation between eqFP611, Rtms5 and DsRed which to differences in the that with the and of in a new immediate environment of the and of the chromophore and in the eqFP611 structure the DsRed structure and the Rtms5 structure is in the same in are colored are as are as red The tripeptide sequence of the chromophore in eqFP611 is that in DsRed and Rtms5 is and in GFP is and have side of similar the side of the eqFP611 chromophore was observed to adopt an extended conformation that well with the of the DsRed and Rtms5 Nevertheless, has a side and within a that is by the side of and is in the Rtms5 and DsRed the chromophore in The is between the of the a number of and with the side the in eqFP611, Rtms5 and DsRed that the and are of these with the the orientation of which is between the to eqFP611, the of DsRed and Rtms5 are and the of the ring The conformation is between the DsRed and Rtms5 with and with and as well as to and The group of the eqFP611 which is coplanar with the is in the trans between and with and The ring of which the with the is coplanar with to adopts a orientation with to the The group and The in eqFP611 the side of a that is in DsRed and however, in these the side is from the chromophore b and A to the eqFP611 chromophore was observed that was to an acetate In Rtms5 and DsRed a water at the chromophore to be a of between the at and in the which on the chromophore environment For example, in DsRed, the side of and with and in eqFP611, the to such that a with and a that is to an acetate In is by an the group of as is observed to a similar to that of DsRed b and We have determined the 2.0-Å crystal structure of eqFP611 and Table and to the of DsRed and Rtms5. A of is the conformation of the eqFP611 In the chromophore conformation of eqFP611 with the highly fluorescent DsRed and the weakly fluorescent the is to be in the of the group In eqFP611, ring is in a trans coplanar in DsRed the ring is in a coplanar in Rtms5 the ring is in a trans and of with the In comparison to DsRed, the group is the that Rtms5 is weakly DsRed and eqFP611 are highly the conformations of the in these that of the chromophore is for a fluorescence The fluorescence of DsRed, eqFP611 and Rtms5 are and Lukyanov S. Lukyanov K.A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, 8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar, M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure. 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). A number of to the observed trans coplanar conformation of the eqFP611 the with to a in Rtms5 the group of to the orientation of the ring two of the eqFP611 phenoxy group with on and to be important for the orientation of the ring and In eqFP611, the group with The in Rtms5 is which with the phenoxy The presence of a in Rtms5 to the of the group of the of the of eqFP611 with the phenoxy The in Rtms5 and DsRed are and in DsRed with the phenoxy group in a similar to in eqFP611, in Rtms5 the In to the group to of the of the of the The presence of an within the eqFP611 chromophore extends the system as observed in DsRed and is with the red-shifted emission of the protein. with DsRed the fluorescence emission of eqFP611 is red-shifted by almost nm (8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar), while the emission of Rtms5 (12Prescott M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure. 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) is red-shifted by more Differences in the chromophore conformations are not the these spectral to red emission to arise from the chromophore in eqFP611, DsRed and Rtms5 as the the with their One for the observed in the fluorescence of eqFP611 is the of the chromophore with The ring of the is highly and the of the has been that and of the chromophore are for the red-shifted spectra observed for fluorescent variants of GFP from A. containing at R.M. M.A. Kallio K. Remington Structure. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of with or results in a of the of emission of nm to the GFP R.M. Tsien R.Y. Nature. PubMed Scopus Google Scholar, M. Kallio K. Tsien R.Y. Remington S.J. PubMed Scopus Google Scholar). Other differences in the the chromophore were observed that the fluorescence of eqFP611. For example, differences in the conformation of a at between DsRed and eqFP611 the of the The eqFP611 crystal structure that eqFP611 a 222 tetramer similar to that observed for DsRed and Rtms5. However, eqFP611 was reported to have a to DsRed (8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar). concentrations for a monomeric of eqFP611 was in the highly monomeric DsRed are in eqFP611, for in eqFP611 (8Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (213) Google Scholar). GFP was new variants have been and GFP homologs isolated that have and One has been for the non-fluorescent protein asFP595 isolated from A. protein fluorescent when with green light and has been for in Zaraisky A.G. Lukyanov S. Lukyanov Nat. Biotechnol. 2003; PubMed Scopus Google Scholar). A of the has been in which the in the from the non-fluorescent to fluorescent form of the protein was a trans to of the chromophore Lukyanov S. Lukyanov K.A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The data together with mutagenesis and structural of related proteins such as Rtms5 and eqFP611 a detailed understanding of