A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure (original) (raw)
Related papers
Biochemistry, 1997
The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea Victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 Å, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pK a1 ) 4.9 ( 0.1, pK a2 ) 12.0 ( 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B.
Journal of Biological Chemistry, 2008
The far-red fluorescent protein mKate ( ex , 588 nm; em , 635 nm; chromophore-forming triad Met 63 -Tyr 64 -Gly 65 ), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 Å . The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser 143 , Leu 174 , and Arg 197 residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val 93 , Arg 122 , Glu 155 , Arg 157 , Asp 159 , His 169 , Ile 171 , Asn 173 , Val 192 , Tyr 194 , and Val 216 , are most likely responsible for the observed monomeric state of the protein in solution.
Proteins: Structure, Function …, 2000
The mutant F99S/M153T/V163A of the Green Fluorescent Protein (c3-GFP) has spectral characteristics similar to the wild-type GFP, but it is 42-fold more fluorescent in vivo. Here, we report the crystal structure and the refolding properties of c3-GFP and compare them with those of the less fluorescent wt-GFP and S65T mutant. The topology and the overall structure of c3-GFP is similar to the wild-type GFP. The three mutated residues, Ser99, Thr153, and Ala163, lie on the surface of the protein in three different -strands. The side chains of Ser99 and Thr153 are exposed to the solvent, whereas that of Ala163 points toward the interior of the protein. No significant deviation from the structure of the wild-type molecule is found around these positions, and there is not clear evidence of any distortion in the position of the chromophore or of the surrounding residues induced by the mutated amino acids. In vitro refolding experiments on ureadenatured c3-GFP reveal a renaturation behavior similar to that of the S65T molecule, with kinetic constants of the same order of magnitude. We conclude that the higher fluorescence activity of c3-GFP can be attributed neither to particular structural features nor to a faster folding process, as previously proposed. Proteins 2000;41:429 -437.
Biochemistry, 2005
The mature self-synthesizing p-hydroxybenzylideneimidazolinone-like fluorophores of Discosoma red fluorescent protein (DsRed) and Aequorea Victoria green fluorescent protein (GFP) are extensively studied as powerful biological markers. Yet, the spontaneous formation of these fluorophores by cyclization, oxidation, and dehydration reactions of tripeptides within their protein environment remains incompletely understood. The mature DsRed fluorophore (Gln 66, Tyr 67, and Gly 68) differs from the GFP fluorophore by an acylimine that results in Gln 66 CR planar geometry and by a Phe 65-Gln 66 cis peptide bond. DsRed green-to-red maturation includes a green-fluorescing immature chromophore and requires a chromophore peptide bond trans-cis isomerization that is slow and incomplete. To clarify the unique structural chemistry for the individual immature "green" and mature "red" chromophores of DsRed, we report here the determination and analysis of crystal structures for the wild-type protein (1.4 Å resolution), the entirely green DsRed K70M mutant protein (1.9 Å resolution), and the DsRed designed mutant Q66M (1.9 Å resolution), which shows increased red chromophore relative to the wild-type DsRed. Whereas the mature, red-fluorescing chromophore has the expected cis peptide bond and a sp 2 -hybridized Gln 66 CR with planar geometry, the crystal structure of the immature green-fluorescing chromophore of DsRed, presented here for the first time, reveals a trans peptide bond and a sp 3 -hybridized Gln 66 CR with tetrahedral geometry. These results characterize a GFP-like immature green DsRed chromophore structure, reveal distinct mature and immature chromophore environments, and furthermore provide evidence for the coupling of acylimine formation with trans-cis isomerization. . 1 Abbreviations: DsRed, Discosoma red fluorescent protein; FRET, fluorescence resonance energy transfer; GFP, Aequorea Victoria green fluorescent protein; asCP, Anemonia sulcata chromoprotein; KFP, Anemonia sulcata kindling fluorescent protein; eqFP611, Entacmaea quadricolor fluorescent protein; rtms5, Montipora efflorescens fluorescent protein; mRFP1, Discosoma monomeric red fluorescent protein. 9833 Biochemistry 2005, 44, 9833-9840 10.
Photo-Induced Peptide Cleavage in the Green-to-Red Conversion of a Fluorescent Protein
Molecular Cell, 2003
Advanced Technology Development Group Brain Science Institute dene)-5-imidazolinone, by nucleophilic attack of Gly 67 -N␣ on the carbonyl of Ser 65 , dehydration, and oxidation of The Institute of Physical and Chemical Science (RIKEN) 2-1 Hirosawa, Wako-city the ␣- bond in Tyr 66 (Heim et al., 1994; Tsien, 1998; Reid and Flynn, 1997). One example of GFP-like protein Saitama 351-0198 Japan is a red-emitting fluorescent protein, DsRed (Matz et al., 1999). DsRed fluoresces first green and then red, implying the existence of some modification of chromophore structure during its maturation (Baird et al., 2000; of Medical Biophysics University of Toronto Mizuno et al., 2001). Recent structural studies have shown that a tripeptide in DsRed (Gln 66 -Tyr 67 -Gly 68 ) anal-Toronto, Ontario M5G 2M9 Canada ogous to the chromophore-forming sequence in Aequorea GFP forms the same structure, 4-(p-hydroxyben-3 Department of Biomolecular Science Toho University zylidene)-5-imidazolinone, and that the C␣-N␣ bond of Gln 66 then oxidizes as the protein matures (Figure 1B) 2-2-1 Miyama, Funabashi Chiba 274-8510 (Gross et al., 2000; Wall et al., 2000; Yarbrough et al., 2001). Japan Kaede is a recently cloned fluorescent protein from a stony coral, Trachyphyllia geoffroyi (Ando et al., 2002).
Structure, 2004
goes a fully reversible, light-induced modification of its chromophore . PYP is a bacterial photoreceptor initially isolated from Halorhodospira halophila (Meyer, 1985), which is believed to be involved in a negative phototactic response to blue light . After absorbing a blue light photon, the covalently attached 3 Consortium for Advanced Radiation Sources University of Chicago coumaric acid chromophore undergoes trans to cis isomerization, which initiates a fully reversible photocy-Chicago, Illinois 60637 4 National Institutes of Health cle that lasts on the order of 1 s . The photon energy is transduced into a structural signal as the mole-Bethesda, Maryland 20892 5 European Synchrotron Radiation Facility cule thermally relaxes through a series of spectroscopically distinguishable intermediates, in which the final Grenoble Cedex 9 France two intermediates are denoted pR and pB (alternatively, I1 and I2). The lifetimes of successive intermediates progressively increase throughout the photocycle, with that of pR being 052ف s and of the final intermediate, Summary pB, 051ف ms (Hoff et al., 1994). Although these intermediates are spectroscopically homogeneous, biphasic
Structure and single crystal spectroscopy of Green Fluorescent Proteins
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2011
Usually, spectroscopic data on proteins in solution are interpreted at molecular level on the basis of the threedimensional structures determined in the crystalline state. While it is widely recognized that the protein crystal structures are reliable models for the solution 3D structures, nevertheless it is also clear that sometimes the crystallization process can introduce some "artifacts" that can make difficult or even flaw the attempt to correlate the properties in solution with those in the crystalline state. In general, therefore, it would be desirable to identify some sort of control. In the case of the spectroscopic properties of proteins, the most straightforward check is to acquire data not only in solution but also on the crystals. In this regard, the Green Fluorescent Protein (GFP) is an interesting case in that a massive quantity of data correlating the spectroscopic properties in solution with the structural information in the crystalline state is available in literature. Despite that, a relatively limited amount of spectroscopic studies on single crystals of GFP or related FPs have been described. Here we review and discuss the main spectroscopic (in solution) and structural (in crystals) studies performed on the GFP and related fluorescent proteins, together with the spectroscopic analyses on various FPs members in the crystalline state. One main conclusion is that "in cristallo" spectroscopic studies are useful in providing new opportunities for gathering information not available in solution and are highly recommended to reliably correlate solution properties with structural features. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Subatomic resolution X-ray structures of green fluorescent protein
IUCrJ, 2019
Green fluorescent protein (GFP) is a light-emitting protein that does not require a prosthetic group for its fluorescent activity. As such, GFP has become indispensable as a molecular tool in molecular biology. Nonetheless, there has been no subatomic elucidation of the GFP structure owing to the structural polymorphism around the chromophore. Here, subatomic resolution X-ray structures of GFP without the structural polymorphism are reported. The positions of H atoms, hydrogen-bonding network patterns and accurate geometric parameters were determined for the two protonated forms. Compared with previously determined crystal structures and theoretically optimized structures, the anionic chromophores of the structures represent the authentic resonance state of GFP. In addition, charge-density analysis based on atoms-inmolecules theory and noncovalent interaction analysis highlight weak but substantial interactions between the chromophore and the protein environment. Considered with the derived chemical indicators, the lone pair-interactions between the chromophore and Thr62 should play a sufficient role in maintaining the electronic state of the chromophore. These results not only reveal the fine structural features that are critical to understanding the properties of GFP, but also highlight the limitations of current quantum-chemical calculations. research papers 388 Kiyofumi Takaba et al. Green fluorescent protein IUCrJ (2019). 6, 387-400 research papers IUCrJ (2019). 6, 387-400 Kiyofumi Takaba et al. Green fluorescent protein 389
Backbone Dynamics of Green Fluorescent Protein and the Effect of Histidine 148 Substitution †
Biochemistry, 2003
Green fluorescent protein (GFP) and its mutants have become valuable tools in molecular biology. GFP has been regarded as a very stable and rigid protein with the -barrel shielding the chromophore from the solvent. Here, we report the 15 N nuclear magnetic resonance (NMR) studies on the green fluorescent protein (GFPuv) and its mutant His148Gly. 15 N NMR relaxation studies of GFPuv show that most of the -barrel of GFP is rigid on the picosecond to nanosecond time scale. For several regions, including the first R-helix and -sheets 3, 7, 8, and 10, increased hydrogen-deuterium exchange rates suggest a substantial conformational flexibility on the microsecond to millisecond time scales. Mutation of residue 148 located in -sheet 7 is known to have a strong impact on the fluorescence properties of GFPs. UV absorption and fluorescence spectra in combination with 1 H-15 N NMR spectra indicate that the His148Gly mutation not only reduces the absorption of the anionic chromophore state but also affects the conformational stability, leading to the appearance of doubled backbone amide resonances for a number of residues. This suggests the presence of two conformations in slow exchange on the NMR time scale in this mutant.
FEBS letters, 2018
The mechanism of green fluorescent protein (GFP) chromophore formation is still not clearly defined. Two mechanisms have been proposed: cyclisation-dehydration-oxidation (Mechanism A) and cyclisation-oxidation-dehydration (Mechanism B). To distinguish between these mechanisms, we generated a non-fluorescent mutant of GFP, S65T/G67A-GFP. This mutant folds to a stable, native-like structure but lacks fluorescence due to interruption of the chromophore maturation process. Mass spectrometric analysis of peptides derived from this mutant reveal that chromophore formation follows only mechanism A, but that the final oxidation reaction is suppressed. This result is unexpected within the pool of examined GFP mutants, since for the wild-type GFP, there is strong support for mechanism B.