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Structural features responsible for GFPuv and S147P-GFP’s improved fluorescence
Chemical Physics 310 (2005) 25–31 www.elsevier.com/locate/chemphys
Structural features responsible for GFPuv and S147P-GFPÕs improved fluorescence Nana Yaa Baffour-Awuah, Flavia Fedeles, Marc Zimmer
*
Chemistry Department, Connecticut College, Box 5624, New London, CT 06340, USA Received 6 August 2004; accepted 20 September 2004 Available online 28 October 2004
Abstract Green fluorescent protein (GFP) is used as a biological marker. It is a protein in the jellyfish, Aequorea victorea, which is found in the cold Pacific Northwest. Mature GFP, i.e. fully fluorescent GFP, is most efficiently formed at temperatures well below 37 C. The GFPuv (F99S/M153T/V163A) and S147P-GFP mutants mature more efficiently at room temperature than wild-type GFP, and therefore result in increased fluorescence at room temperature. Computational methods have been used to examine whether the low-energy precyclized forms of these improved GFP-mutants are preorganized so that they can more efficiently form the chromophore than the wild-type and S65T-GFP. All mutations examined (S147P, F99S, M153T, V163A and F99S/M153T/V163A) more efficiently preorganize the immature precyclized forms of GFP for chromophore formation than immature wild-type GFP. It has been proposed that Arg96 is involved in chromophore formation. Our calculations suggest that the M153T and V163A mutations in GFPuv maybe partially responsible for the increased maturation efficiency observed in GFPuv because they improve the Arg96– Tyr66 interaction. The same is true for the S147P mutation in S147P-GFP. 2004 Elsevier B.V. All rights reserved. Keywords: Chromophore formation; Cycle3; Folding mutants; Green fluorescent protein; Thermostability
1. Introduction Green fluorescent protein (GFP) is widely used as a biological marker. It is particularly useful due to its stability, and the fact that its chromophore is formed in an autocatalytic cyclization that does not require a cofactor. This has enabled researchers to use GFP as a tracer in living systems and it has led to GFPÕs wide spread use in cell dynamics and development studies. Furthermore, it appears that fusion of GFP to a protein does not alter the function or location of the protein [1,2]. GFP is a protein in the jellyfish, Aequorea victorea, which is found in the cold Pacific Northwest [3]. The autocatalytic chromophore formation of 65Ser–Tyr– *
Corresponding author. Tel.: +1 86 043 92476; fax: +1 86 043 92477. E-mail address: [email protected] (M. Zimmer). 0301-0104/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.chemphys.2004.09.031
Gly67, Scheme 1, is comprised of three steps: formation of the five-membered imidazole ring by nucleophilic attack of the Gly67 amide nitrogen on the Ser65 carbonyl carbon, dehydration of the Ser65 carbonyl oxygen, and oxidation of the Ca–Cb bond to form the conjugated chromophore [4–6]. The initial precyclized GFP is referred to as immature GFP and is non-fluorescent. Two different mechanisms have been proposed for the chromophore maturation: a cyclization–oxidation–dehydration mechanism [7] and a conjugation trapping mechanism [8]. Mature GFP, i.e. fully fluorescent GFP, is most efficiently formed at temperatures well below 37 C. This has limited the uses of GFP, especially in mammalian cells, and has led to the search for GFP mutants that mature more efficiently at higher temperatures. Two mutants that are much less thermosensitive than wild-type GFP are S147P-GFP and GFPuv (also known as
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N.Y. Baffour-Awuah et al. / Chemical Physics 310 (2005) 25–31 O
Gly67
O
O
Tyr66 N
..
HN HO
Cyclization O
N
Autocatalytic N
Oxidation
HO
N
Chromophore N HO
Ser65 Immature precyclized GFP
Mature Fluorescent GFP
Scheme 1.
Cycle3). GFPuv is a F99S/M153T/V163A mutant [9], all the mutations are located on the surface of the protein, see Fig. 1. It has a whole cell fluorescence signal that is 45 times greater than that produced by wild-type GFP [9,10], and it does not loose any maturation efficiency if it is expressed at 37 C rather than 28 C [11]. The expression level in Escherichia coli of wtGFP and GFPuv are the same, at about 75% of total protein. The reasons for its thermostability are not know, but it has been suggested that it might be due to improved folding, reduced aggregation or improved chromophore formation. The S147P mutant was found by low fidelity PCR amplification of GFP cDNA, and then added to the
commonly used S65T mutation [12], we will refer to this as S147P-GFP. The resultant mutant was found to be sixfold brighter than S65T-GFP. Furthermore, the S147P-GFP mutant only lost 45% of its fluorescent intensity when it was incubated at 37 C rather than at 23 C, while S65T-GFP lost 90% of its fluorescent intensity when incubated at the higher temperature [12]. In this paper, we will refer to GFP mutants that are more efficient than wt-GFP at becoming fluorescent when expressed at temperatures higher than 30 C as thermostable mutants. It has been shown that the reason for the low thermostability of wild-type GFP is the inhibition of chromophore formation, and not a reduction in fluorescence of the pre-existing properly matured chromophore [13]. In fact once the chromophore in GFP has been formed it retains its fluorescence even when heated up to 65 C [4]. Three reasons for wt-GFPÕs low thermostability that have been proposed in the literature are; ineffective folding; aggregation of GFP molecules; and incomplete chromophore formation. Numerous studies have disproven the idea that inefficient folding is the cause for the reduced thermostability observed in GFP [10,14]. However, to-date no investigations of improved chromophore formation in thermostable mutants have been published – in this paper, we therefore report the results of a computational investigation designed to determine the structural consequences of the F99S/M153T/ V163A (GFPuv) and S147P mutations on the immature precyclized GFP intermediate.
2. Methods
Fig. 1. Residue 147 (green CPK) occurs at the beginning of the only break between the 11 b-sheets of GFPÕs b-barrel structure. The chromophore (tube representation) occurs in the center of the barrel, while all the residues that are mutated in GFPuv (shown in red CPK) are located on the surface of the protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The coordinates for the crystal structure of S65TGFP (1EMG.pdb [15]) and GFPuv (1B9C.pdb [14]) were obtained from the Protein Data Bank. Maestro v5.0 and MacroModel v8.0 [16], which was released in 2002, were used to graphically replace the posttranslationally formed chromophore with the precyclized amino acid sequence, henceforth referred to as the immature form. Hydrogen atoms were added to both protein atoms and solvent atoms as required. All calculations were done with the AMBER* forcefield in MacroModel v8.0 using an aqueous GB/SA sol˚ ‘‘hot’’ sphere from the mutated vent model [17]. An 8 A residues (S147P, F99S, M153T, V163A or F99S/
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M153T/V163A) and the chromophore (residues 65, 66 and 67) with secondary constrained spheres of 2 ˚ ) and 2 A ˚ (k = 200 kJ/mol) were used in (k = 100 kJ/A all molecular dynamics simulations, large scale low mode searches [18,19], and Monte Carlo dihedral and molecular positional conformational searches [20,21] performed on the protein. During the Monte Carlo dihedral and molecular positional conformational searches the backbone and side chain torsion angles of the residues flanking the mutated residues and residues 65–68 were randomly varied between 0 and 180. The N–Ca backbone bonds of residues, two residues before and after the mutation being examined, as well as residues 64 and 69 were designated as closure bonds with minimum and maximum closure ˚ , respectively. The side chain tordistances of 1 and 5 A ˚ sion angles of all residues located in a sphere of 8 A around the mutated residue were also randomly varied between 0 and 180. All crystallographically deter˚ hot sphere were mined water molecules within the 8 A ˚ and rotated randomly moved by between 1 and 5 A by between 0 and 180. The chirality of all backbone carbons was maintained. All conformations within 50 kJ/mol of the global minimum were saved. The Monte Carlo conformational searches consisted of 10,000 MC steps with 500 iterations per step. Structures were considered to be unique when a pair of related atoms was ˚ after a least squares separated by more than 0.025 A superposition of all non-hydrogen atoms. The structures obtained by the Monte Carlo searches of the immature S147P-GFP were analyzed using the cluster analysis program Xcluster [22]. Proximity matrices were obtained by determining the pairwise distances between structures, using the rms displacement between pairs of atoms in the first coordination sphere after optimal rigid-body superposition [23,24]. All the carbonyl carbons, carbonyl oxygens, amide nitrogen and amide
27
hydrogens of the main chains of strands 7, 8 and 10, see Fig. 2, were overlapped in the cluster analysis. The molecular dynamics simulations of S147P-GFP ˚ +3 A ˚ +2 A ˚ sphere centered around the used an 8 A S147P mutation and the chromophore. A 30 ps preequilibrium run was started from the fully minimized lowest energy structure found in the Monte Carlo search with an initial temperature of 300 K and time steps of 1.5 fs. The actual simulations were run at 300 K for 3 and 5 ns with 1.5 fs time steps. All molecular dynamics calculations used the AMBER* force field and SHAKE constrained hydrogens. The immature forms of F99S, M153T, V163A and F99S/M153T/V163A GFP were also subjected to a 5000 step large scale low mode conformational search [18,19].
3. Results and discusion It has been shown that thermostable mutants such as GFPuv have an increased fluorescence at higher temperatures due to their more efficient chromophore formation [13]. After being expressed GFP has to undergo a number of steps before it forms the chromophore and becomes fluorescent. The protein has to fold into the correct b-barrel conformation, the autocatalytic cyclization has to occur, and the Ca–Cb bond of Tyr66 needs to be oxidized. The increased fluorescence of GFPuv and S147P-GFP at higher temperatures has to be due to an improvement in one or more of the three steps described above. We have used computational methods to find the low-energy conformations of the fully folded but immature forms of the single point mutations S147P, F99S, M153T, and V163A, as well as the GFPuv mutant (i.e. F99S/M153T/V163A), and they were analyzed to find
Fig. 2. Schematic representation of the occupancy of backbone–backbone H-bonds of the GFP b-barrel during a 1 ns simulation of GFP wild-type with a neutral chromophore. Thick lines indicate well-preserved H-bonds with occupancies higher than 80%, normal lines indicate H-bonds of intermediate strength (60–80% occupancy), and thin lines indicate weak H-bonds (<60% occupancy) [26]. (Copyright: J. Phys. Chem. B 103 (16) (1999).)
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the possible causes for their increased thermostability. In order to ensure that all residues involved in producing the increased thermostability were considered in ˚ from all the residues our calculations a hot-sphere of 8 A in the chromophore and the mutated residues was used. It is impossible to do a thorough conformational search on the whole protein using currently available supercomputers/Beowulf clusters. There are 53 crystal structures of GFP and GFP-like proteins in the protein data bank [25]. They all have 11 stranded b-barrels with a central chromophore connected to the barrel by two a helices. In all GFP mutants there is very little structural variation, except around the mutation site. Therefore, ˚ we felt confident to limit our calculation to an 8 A sphere from the chromophore forming and mutated residues. In the case of the S147P-GFP mutant a little more than half the protein is in the hot-sphere, while more than 2/3 of the protein is included in the simulations of GFPuv (F99S/M153T/V163A). Experiments have shown that there is no difference in the folding of GFP and GFPuv [10,14], therefore its unlikely that the folding of GFP into its characteristic b-barrel is responsible for its thermostability. The same experiments have not been done with the S147P-GFP mutant. Therefore, it is quite possible that the S147P mutation might improve folding. Unfortunately, there are no currently available computational methods to evaluate the folding ability of proteins. However, the S147P mutation is at the beginning of the only imperfection in the 11 strand b-barrel, Fig. 1, and residues 143–147 are the only residues in the 11 b-strands that did not form well-preserved hydrogen bonds in a molecular dynamics study [26] (Fig. 2). Although introduction of proline in the middle of the antiparallel b-sheet results in the loss of one potential hydrogen-bond donor, we examined the possibility that the S147P mutation might improve backbone–backbone hydrogen bonding between residues 143–146 and the neighboring b-sheets, which in turn could be responsible for improved folding. A 10,000 step Monte Carlo dihedral and molecular positional conformational search of immature S147PGFP found 551 conformations that were found within 50 kJ/mol of the lowest energy conformation of the S147P-GFP mutant. A cluster analysis was undertaken to establish whether the S147P mutation results in low-energy conformations with improved hydrogen bonding between the backbones of the strands 7 and 10, as well between strands 7 and 8. The aim of a cluster analysis is to place objects into groups, also called clusters, in such a way that all the objects within a cluster are very similar and that all the objects in different clusters are very dissimilar to each other. In this study the Xcluster program [22], which is an agglomerative, hierarchical, single-link method, was used. All the carbonyl carbons, carbonyl oxygens, amide
nitrogen and amide hydrogens of strands 7, 8 and 10, see Fig. 2, were superimposed and the rms displacement between their positions in the different clusters was used as a clustering criteria. The separation ratio [22], distance maps and mosaics were used to find nine conformation groups (clusters). The lowest energy conformations, average conformations and representative conformations from each cluster were examined for improved backbone–backbone hydrogen bonding. No indications of improved backbone–backbone hydrogen bonding were found, nor was any evidence found for improved hydrogen bonding between the amino acid sidechains of residues 143–147 with residues from strand 8 and 10. 3.1. Autocatalytic cyclization The most obvious conformational prerequisite for the cyclization step, shown in Scheme 1, is that the amide nitrogen of Gly67 and the carbonyl carbon of Ser65 have to be in close proximity to each other so that the nucleophilic attack shown can occur. The conformation which preorganizes Ser65–Gly67 for cyclization has been termed the tight-turn conformation [27]. Earlier calculations have shown that in immature wild-type GFP the distance between the amide nitrogen of Gly67 and the carbonyl carbon of Ser65 is shorter than ˚ ) [27]. The same the sum of their covalent radii (3.25 A tight turn has been observed in the recently solved crystal structures of the precyclized forms of the S65GY66G and R96A GFP mutants, which have distances ˚ , respectively [8]. of 3.0 and 3.2 A In order to establish whether the S147P mutation aids in chromophore formation Monte Carlo dihedral and molecular positional conformational (MC) searches of the immature S147P-GFP and S65T-GFP were undertaken. The S65T-GFP [15] was used as a baseline in place of wild-type GFP because the S147P-GFP contains the S65T mutation. The distribution of the intramolecular distances between the carbonyl carbon of Thr65 and the amide nitrogen of Gly67 was determined from the resultant low-energy conformations. The average distance between the amide nitrogen of Gly67 residue and the carbonyl carbon of Ser65 residue for the ˚ , smaller than the immature S147P-GFP was 3.00 A comparable average distance for the immature S65T˚ ). Also, in the case of the S147P variant, GFP (3.15 A ˚, all the structures had a distance lower than 3.2 A ˚ for the whereas the distance ranged from 3 to 4.1 A S65T-GFP. Based on this distribution we conclude that immature S147P-GFP has tighter turn conformations than S65T-GFP. All the low-energy S147P-GFP conformations have proline147 in a trans conformation. A MC search with proline 147 constrained in a cis conformation found no low-energy conformations within 50 kJ/mol of the lowest trans Pro147 conformations.
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Room temperature molecular dynamics simulations of the immature forms of S65T-GFP and S147P-GFP showed similar behavior to that observed in the Monte Carlo dihedral and molecular positional conformational (MC) searches. During a 5 ns simulation the intramolecular distances between the carbonyl carbon of Thr65 ˚ for and the amide nitrogen of Gly67 averages 3.38 A S65T-GFP, while the S147P-GFP is preorganized in a tighter turn conformation with an average distance of ˚ . For S65T-GFP there was no low-energy confor2.94 A ˚ , whereas for mation with a distance smaller than 3.0 A ˚. the S147P variant the lowest distance was 2.725 A Recent DFT calculations seem to suggest that the oxidation of Tyr66 to dehydroTyr66 may occur prior to cyclization [28,29]. Therefore, Monte Carlo conformational searches of immature dehydro S65T-GFP and immature dehydro S147P-GFP were conducted. Dehydration of Tyr66 reduces the flexibility of the chromophore forming region and preorganizes wild-type for chromophore formation. The S147P mutation already preorganizes GFP for chromophore formation, oxidation of Tyr66 does not further preorganize residues 65–67. However, it does limit the number of low-energy conformations available to dehydro S147P-GFP. GFPuv has three mutations, which are responsible for its increased ‘‘thermostability’’, F99S/M153T/ V163A. They are all located on the surface of the protein, see Fig. 1. Monte Carlo dihedral and molecular positional conformational searches identical to those described for S147P-GFP were carried out with immature F99S, M153T, V163A and F99S/M153T/V163A GFPmutants. Fig. 3 is a histogram summarizing the results of the searches. The F99S, M153T and V163A single mutants all have significantly shorter carbonyl carbonamide nitrogen distances than that found in S65TGFP. The combined effect of the three mutations is a GFPuv mutant that is distinctly preorganized for cycli-
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zation. The distance between the carbonyl carbon of Ser65 and the amide nitrogen of Gly67 is significantly shorter in all the low-energy conformations found for immature GFPuv than for those found for S65T-GFP and even S147P-GFP. Therefore, it seems that both the S147P mutation and the combination of the three F99S/M153T/V163A mutations found in GFPuv aid in forming a tighter-turn conformation than that found for S65T-GFP, which should result in more efficient chromophore formation. Before the crystal structure of GFP was solved we proposed that a tight-turn conformation in immature GFP was not the only requirement for an autocatalytic cyclization such as that observed in GFP. We suggested that an arginine is required to activate the carbonyl carbon for nucleophilic attack by the amide nitrogen [30]. The crystal structures of GFP revealed that Arg96 was in close proximity of the chromophore [31,32]. In fact it is found to be hydrogen-bonded to the carbonyl oxygen of the five-membered imidazole ring in all crystal structures of GFP and its analogs [7]. It has also been found that Arg96 is conserved in all GFPÕs and GFP analogs [33]. However, it is not essential for chromophore formation. Getzoff and co-workers [8] have shown that R96A mutants slow the cyclization reaction from minutes to months. They propose that Arg96 contributes to the architectural distortions important for peptide cyclization and increases the nucleophilicity of the attacking Gly67 nitrogen, see Fig. 4. We have therefore examined the distance between Arg96 and the carbonyl oxygen of Tyr66 in the S147P, F99S, M153T, V163A and F99S/M153T/V163A precyclized structures to establish whether any of the mutants are less thermally sensitive due to improved Arg96–Tyr66 interactions. The immature low-energy ˚ ), S147P conformations of V163A (average = 2.65 A ˚ ), M153T (average = 2.70 A ˚ ) and (average = 2.60 A
Fig. 3. The distribution of the intramolecular distances between the carbonyl carbon of residue65 and the amide nitrogen of Gly67 (see Scheme 1) for the immature forms of the S147P, F99S, M153T, V163A and F99S/M153T/V163A GFP mutants. The structures were obtained by the Monte Carlo dihedral and molecular positional conformational searches described in Section 2.
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N.Y. Baffour-Awuah et al. / Chemical Physics 310 (2005) 25–31 R
H N
C
H
N H
Arg96 + H
-O
Gly67
Tyr66
R N H N O
Ser65 R
OH
HOH Fig. 4. Arg96 has been proposed to enhance chromophore by nucleophilic activation of the amide nitrogen of Tyr66 [8].
˚ ) all have shorter distances beGFPuv (average = 2.60 A tween Arg96 and the carbonyl oxygen of Tyr66 than the ˚ found in the matured S65T-GFP distance of 2.81 A structure (pdb code = 1emg.pdb), while the F99S (aver˚ ) distance is significantly longer. age distance = 3.69 A Therefore, if one can assume that an increased Arg96– Tyr66 interaction, which is reflected in a decreased Arg96–Tyr66 distance, can lead to more efficient chromophore formation, then our calculations suggest that the M153T and V163A mutations in GFPuv are partially responsible for the increased maturation efficiency observed in GFPuv. The same is true for the S147P mutation in S147P-GFP. Although Glu222 is conserved in all GFP-like proteins it has been shown that it is not essential in generating the chromophore [34,35]. However, both the conjugation trapping mechanism [8] and the proton transfer mechanism [29] have a role for Glu222 in chromophore formation, therefore we have measured all the Glu222–Ser65 distances in all our immature low-energy structures to establish whether Glu222Õs increased role in chromophore formation might be responsible for the decreased thermosensitivity of the mutants we have studied. The low-energy conformations of the F99S ˚ ) are the only strucmutation (average distance = 3.98 A tures that had significantly shorter Glu222–Ser65 distances than those observed in the low-energy conformations obtained for immature S65T-GFP (aver˚ ). age distance = 5.42 A 3.2. Chromophore oxidation Not much is known about the chromophore oxidation step in the chromophore forming step. In most commonly accepted mechanisms the oxidation step follows the autocyclization [4,7,8], and is presumed to be the slow step in chromophore formation [6]. However, according to quantum mechanical calculations [28,29]
the oxidation precedes cyclization. The conjugationtrapping mechanism does not address the oxidation of the tyrosine sidechain. In the cyclization–oxidation–dehydration mechanism [7] it has been proposed that the Ca–Cb double bond of Tyr66 is formed by a dehydration reaction utilizing a proton shuttle formed via hydrogenbonded water bridges in which none of the surrounding amino acids having a specific role. If this is how the chromophore oxidation occurs it would be very difficult, with currently available computational techniques and the current knowledge of the mechanism, to use computational methods to find out whether the thermostable mutants are more efficient at chromophore oxidation. We have therefore not been able to use computational methods to establish whether the GFPuv and S147P mutations aid in the oxidation of the chromophore.
4. Conclusion The improved fluorescence of S147P-GFP and GFPuv is due to the fact that these GFP variants are more efficient at forming the chromophore than wild-type GFP, especially at higher temperatures. Initially, it was thought that GFPuv was better than wild-type GFP at folding into the correct b-barrel conformation, and that this led to improved chromophore formation. However, numerous experiments have shown that this is not the case. We have used computational techniques to determine the low-energy conformations of the precyclized forms of S147P-GFP and GFPuv. These structures have been used to show that the F99S, M153T, V163A and S147P mutations may have numerous conformational consequences that could lead to improved chromophore formation. Namely: • All three mutations (F99S, M153T, and V163A) found in GFPuv are responsible for precyclized GFPuv forming a tighter-turn than wild-type and S65T-GFPÕs. The S147P mutation also results in a decreased intramolecular distance between the carbonyl carbon of Thr65 and the amide nitrogen of Gly67 relative to that observed in the precyclized structures obtained for S65T-GFP. • If one can assume that an increased Arg96–Tyr66 interaction, which is reflected in a decreased Arg96– Tyr66 distance, can lead to more efficient chromophore formation, then our calculations suggest that the M153T and V163A mutations in GFPuv are partially responsible for the increased maturation efficiency observed in GFPuv by improving the Arg96– Tyr66 interactions. The same is true for the S147P mutation in S147P-GFP. Although the mutations in GFPuv and S147P may improve the thermosensitivity of the thermostable
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GFP mutants for the reasons suggested above, our calculations do not rule out the possibility that other reasons, such as the effects of the mutations on chromophore oxidation, might be responsible for the observed thermostability.
Acknowledgements M.Z. is a Henry Dreyfus Teacher-Scholar. The NIH and Research Corporation provided support for this research. This project was supported in part by NSF Grant CHE-0116435 as part of the MERCURY supercomputer consortium (http://mars.hamilton.edu).
References [1] M. Zimmer, Chem. Rev. 102 (2002) 759. [2] R.Y. Tsien, Annu. Rev. Biochem. 67 (1998) 509. [3] M. Zimmer, Glowing Genes: A Revolution in Biotechnology, Prometheus Books, Amherst, NY, 2005. [4] A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, R.Y. Tsien, TIBS 20 (1995) 448. [5] R. Heim, D.C. Prasher, R.Y. Tsien, Proc. Natl. Acad. Sci. USA 91 (1994) 12501. [6] B.G. Reid, G.C. Flynn, Biochemistry 36 (1997) 6786. [7] M.A. Rosenow, H.A. Huffman, M.E. Phail, R.M. Wachter, Biochemistry 43 (2004) 4464. [8] D.P. Barondeau, C.D. Putnam, C.J. Kassmann, J.A. Tainer, E.D. Getzoff, Proc. Natl. Acad. Sci. USA 100 (2003) 12111. [9] A. Crameri, E.A. Whitehorn, E. Tate, W.P.C. Stemmer, Nat. Biotechnol. 14 (1996) 315. [10] H. Fukuda, M. Arai, K. Kuwajima, Biochemistry 39 (2000) 12025. [11] G.H. Patterson, S.M. Knobel, W.D. Sharif, S.R. Kain, D.W. Piston, Biophys. J. 73 (1997) 2782. [12] Y. Kimata, M. Iwaki, C.R. Lim, K. Kohno, Biochem. Biophys. Res. Commun. 232 (1997) 69.
31
[13] C.R. Lim, Y. Kimata, M. Oka, K. Nomaguchi, K. Kohno, J. Biochem. 118 (1995) 13. [14] R. Battistutta, A. Negro, G. Zanotti, Proteins 41 (2000) 429. [15] M.A. Elsliger, R.M. Wachter, G.T. Hanson, K. Kallio, S.J. Remington, Biochemistry 38 (1999) 5296. [16] F. Mohamadi, N. Richards, W. Guida, R. Liskamp, M. Lipton, C. Caulfield, G. Chang, T. Hendrickson, W. Still, J. Comput. Chem. 11 (1990) 440. [17] W. Still, A. Tempczyk, R. Hawley, T. Hendrickson, J. Am. Chem. Soc. 112 (1990) 6127. [18] G.M. Keseru, I. Kolossvary, J. Am. Chem. Soc. 123 (2001) 12708. [19] I. Kolossvary, G.M. Keseru, J. Comput. Chem. 22 (2001) 21. [20] F. Guarnieri, W. Still, J. Comput. Chem. 15 (1994) 1302. [21] H. Senderowitz, F. Guarnieri, W. Still, J. Am. Chem. Soc. 117 (1995) 8211. [22] P. Shenkin, Q. McDonald, J. Comput. Chem. 15 (1994) 899. [23] W. Kabsch, Acta Crystallogr., Sect. A 32 (1976) 922. [24] W. Kabsch, Acta Crystallogr., Sect. A 34 (1978) 827. [25] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, Nucleic Acids Res. 28 (2000) 235. [26] V. Helms, T.P. Straatsma, J.A. McCammon, J. Phys. Chem. B 103 (1999) 3263. [27] B.R. Branchini, A.R. Nemser, M. Zimmer, J. Am. Chem. Soc. 120 (1998) 1. [28] M. Donnelly, F. Fedeles, M. Wirstam, P.E.M. Siegbahn, M. Zimmer, J. Am. Chem. Soc. 123 (2001) 4679. [29] P.E.M. Siegbahn, M. Wirstam, M. Zimmer, Int. J. Quantum Chem. 81 (2001) 169. [30] M. Zimmer, B.R. Branchini, J.O. Lusins, in: J.W. Hastings, L.J. Kricka, P.E. Stanley (Eds.), Bioluminescence and Chemiluminescence: Proceedings of the 9th International Symposium 1996, Wiley, Chichester, 1996, p. 407. [31] M. Ormoe, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, Science 273 (1996) 1392. [32] F. Yang, L. Moss, G. Phillips, Nat. Biotechnol. 14 (1996) 1246. [33] M.V. Matz, A.F. Fradkov, Y.A. Labas, A.P. Savitisky, A.G. Zaraisky, M.L. Markelov, S.A. Lukyanov, Nat. Biotechnol. 17 (1999) 969. [34] T. Ehrig, D.J. OÕKane, F.G. Pendergast, FEBS Lett. 367 (1995) 163. [35] J. Wiehler, G. Jung, C. Seebacher, Z. Zumbusch, B. Steipe, Chembiochem 4 (2003) 1164.
Structural features responsible for GFPuv and S147P-GFPÕs improved fluorescence Nana Yaa Baffour-Awuah, Flavia Fedeles, Marc Zimmer
*
Chemistry Department, Connecticut College, Box 5624, New London, CT 06340, USA Received 6 August 2004; accepted 20 September 2004 Available online 28 October 2004
Abstract Green fluorescent protein (GFP) is used as a biological marker. It is a protein in the jellyfish, Aequorea victorea, which is found in the cold Pacific Northwest. Mature GFP, i.e. fully fluorescent GFP, is most efficiently formed at temperatures well below 37 C. The GFPuv (F99S/M153T/V163A) and S147P-GFP mutants mature more efficiently at room temperature than wild-type GFP, and therefore result in increased fluorescence at room temperature. Computational methods have been used to examine whether the low-energy precyclized forms of these improved GFP-mutants are preorganized so that they can more efficiently form the chromophore than the wild-type and S65T-GFP. All mutations examined (S147P, F99S, M153T, V163A and F99S/M153T/V163A) more efficiently preorganize the immature precyclized forms of GFP for chromophore formation than immature wild-type GFP. It has been proposed that Arg96 is involved in chromophore formation. Our calculations suggest that the M153T and V163A mutations in GFPuv maybe partially responsible for the increased maturation efficiency observed in GFPuv because they improve the Arg96– Tyr66 interaction. The same is true for the S147P mutation in S147P-GFP. 2004 Elsevier B.V. All rights reserved. Keywords: Chromophore formation; Cycle3; Folding mutants; Green fluorescent protein; Thermostability
1. Introduction Green fluorescent protein (GFP) is widely used as a biological marker. It is particularly useful due to its stability, and the fact that its chromophore is formed in an autocatalytic cyclization that does not require a cofactor. This has enabled researchers to use GFP as a tracer in living systems and it has led to GFPÕs wide spread use in cell dynamics and development studies. Furthermore, it appears that fusion of GFP to a protein does not alter the function or location of the protein [1,2]. GFP is a protein in the jellyfish, Aequorea victorea, which is found in the cold Pacific Northwest [3]. The autocatalytic chromophore formation of 65Ser–Tyr– *
Corresponding author. Tel.: +1 86 043 92476; fax: +1 86 043 92477. E-mail address: [email protected] (M. Zimmer). 0301-0104/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.chemphys.2004.09.031
Gly67, Scheme 1, is comprised of three steps: formation of the five-membered imidazole ring by nucleophilic attack of the Gly67 amide nitrogen on the Ser65 carbonyl carbon, dehydration of the Ser65 carbonyl oxygen, and oxidation of the Ca–Cb bond to form the conjugated chromophore [4–6]. The initial precyclized GFP is referred to as immature GFP and is non-fluorescent. Two different mechanisms have been proposed for the chromophore maturation: a cyclization–oxidation–dehydration mechanism [7] and a conjugation trapping mechanism [8]. Mature GFP, i.e. fully fluorescent GFP, is most efficiently formed at temperatures well below 37 C. This has limited the uses of GFP, especially in mammalian cells, and has led to the search for GFP mutants that mature more efficiently at higher temperatures. Two mutants that are much less thermosensitive than wild-type GFP are S147P-GFP and GFPuv (also known as
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N.Y. Baffour-Awuah et al. / Chemical Physics 310 (2005) 25–31 O
Gly67
O
O
Tyr66 N
..
HN HO
Cyclization O
N
Autocatalytic N
Oxidation
HO
N
Chromophore N HO
Ser65 Immature precyclized GFP
Mature Fluorescent GFP
Scheme 1.
Cycle3). GFPuv is a F99S/M153T/V163A mutant [9], all the mutations are located on the surface of the protein, see Fig. 1. It has a whole cell fluorescence signal that is 45 times greater than that produced by wild-type GFP [9,10], and it does not loose any maturation efficiency if it is expressed at 37 C rather than 28 C [11]. The expression level in Escherichia coli of wtGFP and GFPuv are the same, at about 75% of total protein. The reasons for its thermostability are not know, but it has been suggested that it might be due to improved folding, reduced aggregation or improved chromophore formation. The S147P mutant was found by low fidelity PCR amplification of GFP cDNA, and then added to the
commonly used S65T mutation [12], we will refer to this as S147P-GFP. The resultant mutant was found to be sixfold brighter than S65T-GFP. Furthermore, the S147P-GFP mutant only lost 45% of its fluorescent intensity when it was incubated at 37 C rather than at 23 C, while S65T-GFP lost 90% of its fluorescent intensity when incubated at the higher temperature [12]. In this paper, we will refer to GFP mutants that are more efficient than wt-GFP at becoming fluorescent when expressed at temperatures higher than 30 C as thermostable mutants. It has been shown that the reason for the low thermostability of wild-type GFP is the inhibition of chromophore formation, and not a reduction in fluorescence of the pre-existing properly matured chromophore [13]. In fact once the chromophore in GFP has been formed it retains its fluorescence even when heated up to 65 C [4]. Three reasons for wt-GFPÕs low thermostability that have been proposed in the literature are; ineffective folding; aggregation of GFP molecules; and incomplete chromophore formation. Numerous studies have disproven the idea that inefficient folding is the cause for the reduced thermostability observed in GFP [10,14]. However, to-date no investigations of improved chromophore formation in thermostable mutants have been published – in this paper, we therefore report the results of a computational investigation designed to determine the structural consequences of the F99S/M153T/ V163A (GFPuv) and S147P mutations on the immature precyclized GFP intermediate.
2. Methods
Fig. 1. Residue 147 (green CPK) occurs at the beginning of the only break between the 11 b-sheets of GFPÕs b-barrel structure. The chromophore (tube representation) occurs in the center of the barrel, while all the residues that are mutated in GFPuv (shown in red CPK) are located on the surface of the protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The coordinates for the crystal structure of S65TGFP (1EMG.pdb [15]) and GFPuv (1B9C.pdb [14]) were obtained from the Protein Data Bank. Maestro v5.0 and MacroModel v8.0 [16], which was released in 2002, were used to graphically replace the posttranslationally formed chromophore with the precyclized amino acid sequence, henceforth referred to as the immature form. Hydrogen atoms were added to both protein atoms and solvent atoms as required. All calculations were done with the AMBER* forcefield in MacroModel v8.0 using an aqueous GB/SA sol˚ ‘‘hot’’ sphere from the mutated vent model [17]. An 8 A residues (S147P, F99S, M153T, V163A or F99S/
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M153T/V163A) and the chromophore (residues 65, 66 and 67) with secondary constrained spheres of 2 ˚ ) and 2 A ˚ (k = 200 kJ/mol) were used in (k = 100 kJ/A all molecular dynamics simulations, large scale low mode searches [18,19], and Monte Carlo dihedral and molecular positional conformational searches [20,21] performed on the protein. During the Monte Carlo dihedral and molecular positional conformational searches the backbone and side chain torsion angles of the residues flanking the mutated residues and residues 65–68 were randomly varied between 0 and 180. The N–Ca backbone bonds of residues, two residues before and after the mutation being examined, as well as residues 64 and 69 were designated as closure bonds with minimum and maximum closure ˚ , respectively. The side chain tordistances of 1 and 5 A ˚ sion angles of all residues located in a sphere of 8 A around the mutated residue were also randomly varied between 0 and 180. All crystallographically deter˚ hot sphere were mined water molecules within the 8 A ˚ and rotated randomly moved by between 1 and 5 A by between 0 and 180. The chirality of all backbone carbons was maintained. All conformations within 50 kJ/mol of the global minimum were saved. The Monte Carlo conformational searches consisted of 10,000 MC steps with 500 iterations per step. Structures were considered to be unique when a pair of related atoms was ˚ after a least squares separated by more than 0.025 A superposition of all non-hydrogen atoms. The structures obtained by the Monte Carlo searches of the immature S147P-GFP were analyzed using the cluster analysis program Xcluster [22]. Proximity matrices were obtained by determining the pairwise distances between structures, using the rms displacement between pairs of atoms in the first coordination sphere after optimal rigid-body superposition [23,24]. All the carbonyl carbons, carbonyl oxygens, amide nitrogen and amide
27
hydrogens of the main chains of strands 7, 8 and 10, see Fig. 2, were overlapped in the cluster analysis. The molecular dynamics simulations of S147P-GFP ˚ +3 A ˚ +2 A ˚ sphere centered around the used an 8 A S147P mutation and the chromophore. A 30 ps preequilibrium run was started from the fully minimized lowest energy structure found in the Monte Carlo search with an initial temperature of 300 K and time steps of 1.5 fs. The actual simulations were run at 300 K for 3 and 5 ns with 1.5 fs time steps. All molecular dynamics calculations used the AMBER* force field and SHAKE constrained hydrogens. The immature forms of F99S, M153T, V163A and F99S/M153T/V163A GFP were also subjected to a 5000 step large scale low mode conformational search [18,19].
3. Results and discusion It has been shown that thermostable mutants such as GFPuv have an increased fluorescence at higher temperatures due to their more efficient chromophore formation [13]. After being expressed GFP has to undergo a number of steps before it forms the chromophore and becomes fluorescent. The protein has to fold into the correct b-barrel conformation, the autocatalytic cyclization has to occur, and the Ca–Cb bond of Tyr66 needs to be oxidized. The increased fluorescence of GFPuv and S147P-GFP at higher temperatures has to be due to an improvement in one or more of the three steps described above. We have used computational methods to find the low-energy conformations of the fully folded but immature forms of the single point mutations S147P, F99S, M153T, and V163A, as well as the GFPuv mutant (i.e. F99S/M153T/V163A), and they were analyzed to find
Fig. 2. Schematic representation of the occupancy of backbone–backbone H-bonds of the GFP b-barrel during a 1 ns simulation of GFP wild-type with a neutral chromophore. Thick lines indicate well-preserved H-bonds with occupancies higher than 80%, normal lines indicate H-bonds of intermediate strength (60–80% occupancy), and thin lines indicate weak H-bonds (<60% occupancy) [26]. (Copyright: J. Phys. Chem. B 103 (16) (1999).)
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the possible causes for their increased thermostability. In order to ensure that all residues involved in producing the increased thermostability were considered in ˚ from all the residues our calculations a hot-sphere of 8 A in the chromophore and the mutated residues was used. It is impossible to do a thorough conformational search on the whole protein using currently available supercomputers/Beowulf clusters. There are 53 crystal structures of GFP and GFP-like proteins in the protein data bank [25]. They all have 11 stranded b-barrels with a central chromophore connected to the barrel by two a helices. In all GFP mutants there is very little structural variation, except around the mutation site. Therefore, ˚ we felt confident to limit our calculation to an 8 A sphere from the chromophore forming and mutated residues. In the case of the S147P-GFP mutant a little more than half the protein is in the hot-sphere, while more than 2/3 of the protein is included in the simulations of GFPuv (F99S/M153T/V163A). Experiments have shown that there is no difference in the folding of GFP and GFPuv [10,14], therefore its unlikely that the folding of GFP into its characteristic b-barrel is responsible for its thermostability. The same experiments have not been done with the S147P-GFP mutant. Therefore, it is quite possible that the S147P mutation might improve folding. Unfortunately, there are no currently available computational methods to evaluate the folding ability of proteins. However, the S147P mutation is at the beginning of the only imperfection in the 11 strand b-barrel, Fig. 1, and residues 143–147 are the only residues in the 11 b-strands that did not form well-preserved hydrogen bonds in a molecular dynamics study [26] (Fig. 2). Although introduction of proline in the middle of the antiparallel b-sheet results in the loss of one potential hydrogen-bond donor, we examined the possibility that the S147P mutation might improve backbone–backbone hydrogen bonding between residues 143–146 and the neighboring b-sheets, which in turn could be responsible for improved folding. A 10,000 step Monte Carlo dihedral and molecular positional conformational search of immature S147PGFP found 551 conformations that were found within 50 kJ/mol of the lowest energy conformation of the S147P-GFP mutant. A cluster analysis was undertaken to establish whether the S147P mutation results in low-energy conformations with improved hydrogen bonding between the backbones of the strands 7 and 10, as well between strands 7 and 8. The aim of a cluster analysis is to place objects into groups, also called clusters, in such a way that all the objects within a cluster are very similar and that all the objects in different clusters are very dissimilar to each other. In this study the Xcluster program [22], which is an agglomerative, hierarchical, single-link method, was used. All the carbonyl carbons, carbonyl oxygens, amide
nitrogen and amide hydrogens of strands 7, 8 and 10, see Fig. 2, were superimposed and the rms displacement between their positions in the different clusters was used as a clustering criteria. The separation ratio [22], distance maps and mosaics were used to find nine conformation groups (clusters). The lowest energy conformations, average conformations and representative conformations from each cluster were examined for improved backbone–backbone hydrogen bonding. No indications of improved backbone–backbone hydrogen bonding were found, nor was any evidence found for improved hydrogen bonding between the amino acid sidechains of residues 143–147 with residues from strand 8 and 10. 3.1. Autocatalytic cyclization The most obvious conformational prerequisite for the cyclization step, shown in Scheme 1, is that the amide nitrogen of Gly67 and the carbonyl carbon of Ser65 have to be in close proximity to each other so that the nucleophilic attack shown can occur. The conformation which preorganizes Ser65–Gly67 for cyclization has been termed the tight-turn conformation [27]. Earlier calculations have shown that in immature wild-type GFP the distance between the amide nitrogen of Gly67 and the carbonyl carbon of Ser65 is shorter than ˚ ) [27]. The same the sum of their covalent radii (3.25 A tight turn has been observed in the recently solved crystal structures of the precyclized forms of the S65GY66G and R96A GFP mutants, which have distances ˚ , respectively [8]. of 3.0 and 3.2 A In order to establish whether the S147P mutation aids in chromophore formation Monte Carlo dihedral and molecular positional conformational (MC) searches of the immature S147P-GFP and S65T-GFP were undertaken. The S65T-GFP [15] was used as a baseline in place of wild-type GFP because the S147P-GFP contains the S65T mutation. The distribution of the intramolecular distances between the carbonyl carbon of Thr65 and the amide nitrogen of Gly67 was determined from the resultant low-energy conformations. The average distance between the amide nitrogen of Gly67 residue and the carbonyl carbon of Ser65 residue for the ˚ , smaller than the immature S147P-GFP was 3.00 A comparable average distance for the immature S65T˚ ). Also, in the case of the S147P variant, GFP (3.15 A ˚, all the structures had a distance lower than 3.2 A ˚ for the whereas the distance ranged from 3 to 4.1 A S65T-GFP. Based on this distribution we conclude that immature S147P-GFP has tighter turn conformations than S65T-GFP. All the low-energy S147P-GFP conformations have proline147 in a trans conformation. A MC search with proline 147 constrained in a cis conformation found no low-energy conformations within 50 kJ/mol of the lowest trans Pro147 conformations.
N.Y. Baffour-Awuah et al. / Chemical Physics 310 (2005) 25–31
Room temperature molecular dynamics simulations of the immature forms of S65T-GFP and S147P-GFP showed similar behavior to that observed in the Monte Carlo dihedral and molecular positional conformational (MC) searches. During a 5 ns simulation the intramolecular distances between the carbonyl carbon of Thr65 ˚ for and the amide nitrogen of Gly67 averages 3.38 A S65T-GFP, while the S147P-GFP is preorganized in a tighter turn conformation with an average distance of ˚ . For S65T-GFP there was no low-energy confor2.94 A ˚ , whereas for mation with a distance smaller than 3.0 A ˚. the S147P variant the lowest distance was 2.725 A Recent DFT calculations seem to suggest that the oxidation of Tyr66 to dehydroTyr66 may occur prior to cyclization [28,29]. Therefore, Monte Carlo conformational searches of immature dehydro S65T-GFP and immature dehydro S147P-GFP were conducted. Dehydration of Tyr66 reduces the flexibility of the chromophore forming region and preorganizes wild-type for chromophore formation. The S147P mutation already preorganizes GFP for chromophore formation, oxidation of Tyr66 does not further preorganize residues 65–67. However, it does limit the number of low-energy conformations available to dehydro S147P-GFP. GFPuv has three mutations, which are responsible for its increased ‘‘thermostability’’, F99S/M153T/ V163A. They are all located on the surface of the protein, see Fig. 1. Monte Carlo dihedral and molecular positional conformational searches identical to those described for S147P-GFP were carried out with immature F99S, M153T, V163A and F99S/M153T/V163A GFPmutants. Fig. 3 is a histogram summarizing the results of the searches. The F99S, M153T and V163A single mutants all have significantly shorter carbonyl carbonamide nitrogen distances than that found in S65TGFP. The combined effect of the three mutations is a GFPuv mutant that is distinctly preorganized for cycli-
29
zation. The distance between the carbonyl carbon of Ser65 and the amide nitrogen of Gly67 is significantly shorter in all the low-energy conformations found for immature GFPuv than for those found for S65T-GFP and even S147P-GFP. Therefore, it seems that both the S147P mutation and the combination of the three F99S/M153T/V163A mutations found in GFPuv aid in forming a tighter-turn conformation than that found for S65T-GFP, which should result in more efficient chromophore formation. Before the crystal structure of GFP was solved we proposed that a tight-turn conformation in immature GFP was not the only requirement for an autocatalytic cyclization such as that observed in GFP. We suggested that an arginine is required to activate the carbonyl carbon for nucleophilic attack by the amide nitrogen [30]. The crystal structures of GFP revealed that Arg96 was in close proximity of the chromophore [31,32]. In fact it is found to be hydrogen-bonded to the carbonyl oxygen of the five-membered imidazole ring in all crystal structures of GFP and its analogs [7]. It has also been found that Arg96 is conserved in all GFPÕs and GFP analogs [33]. However, it is not essential for chromophore formation. Getzoff and co-workers [8] have shown that R96A mutants slow the cyclization reaction from minutes to months. They propose that Arg96 contributes to the architectural distortions important for peptide cyclization and increases the nucleophilicity of the attacking Gly67 nitrogen, see Fig. 4. We have therefore examined the distance between Arg96 and the carbonyl oxygen of Tyr66 in the S147P, F99S, M153T, V163A and F99S/M153T/V163A precyclized structures to establish whether any of the mutants are less thermally sensitive due to improved Arg96–Tyr66 interactions. The immature low-energy ˚ ), S147P conformations of V163A (average = 2.65 A ˚ ), M153T (average = 2.70 A ˚ ) and (average = 2.60 A
Fig. 3. The distribution of the intramolecular distances between the carbonyl carbon of residue65 and the amide nitrogen of Gly67 (see Scheme 1) for the immature forms of the S147P, F99S, M153T, V163A and F99S/M153T/V163A GFP mutants. The structures were obtained by the Monte Carlo dihedral and molecular positional conformational searches described in Section 2.
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H N
C
H
N H
Arg96 + H
-O
Gly67
Tyr66
R N H N O
Ser65 R
OH
HOH Fig. 4. Arg96 has been proposed to enhance chromophore by nucleophilic activation of the amide nitrogen of Tyr66 [8].
˚ ) all have shorter distances beGFPuv (average = 2.60 A tween Arg96 and the carbonyl oxygen of Tyr66 than the ˚ found in the matured S65T-GFP distance of 2.81 A structure (pdb code = 1emg.pdb), while the F99S (aver˚ ) distance is significantly longer. age distance = 3.69 A Therefore, if one can assume that an increased Arg96– Tyr66 interaction, which is reflected in a decreased Arg96–Tyr66 distance, can lead to more efficient chromophore formation, then our calculations suggest that the M153T and V163A mutations in GFPuv are partially responsible for the increased maturation efficiency observed in GFPuv. The same is true for the S147P mutation in S147P-GFP. Although Glu222 is conserved in all GFP-like proteins it has been shown that it is not essential in generating the chromophore [34,35]. However, both the conjugation trapping mechanism [8] and the proton transfer mechanism [29] have a role for Glu222 in chromophore formation, therefore we have measured all the Glu222–Ser65 distances in all our immature low-energy structures to establish whether Glu222Õs increased role in chromophore formation might be responsible for the decreased thermosensitivity of the mutants we have studied. The low-energy conformations of the F99S ˚ ) are the only strucmutation (average distance = 3.98 A tures that had significantly shorter Glu222–Ser65 distances than those observed in the low-energy conformations obtained for immature S65T-GFP (aver˚ ). age distance = 5.42 A 3.2. Chromophore oxidation Not much is known about the chromophore oxidation step in the chromophore forming step. In most commonly accepted mechanisms the oxidation step follows the autocyclization [4,7,8], and is presumed to be the slow step in chromophore formation [6]. However, according to quantum mechanical calculations [28,29]
the oxidation precedes cyclization. The conjugationtrapping mechanism does not address the oxidation of the tyrosine sidechain. In the cyclization–oxidation–dehydration mechanism [7] it has been proposed that the Ca–Cb double bond of Tyr66 is formed by a dehydration reaction utilizing a proton shuttle formed via hydrogenbonded water bridges in which none of the surrounding amino acids having a specific role. If this is how the chromophore oxidation occurs it would be very difficult, with currently available computational techniques and the current knowledge of the mechanism, to use computational methods to find out whether the thermostable mutants are more efficient at chromophore oxidation. We have therefore not been able to use computational methods to establish whether the GFPuv and S147P mutations aid in the oxidation of the chromophore.
4. Conclusion The improved fluorescence of S147P-GFP and GFPuv is due to the fact that these GFP variants are more efficient at forming the chromophore than wild-type GFP, especially at higher temperatures. Initially, it was thought that GFPuv was better than wild-type GFP at folding into the correct b-barrel conformation, and that this led to improved chromophore formation. However, numerous experiments have shown that this is not the case. We have used computational techniques to determine the low-energy conformations of the precyclized forms of S147P-GFP and GFPuv. These structures have been used to show that the F99S, M153T, V163A and S147P mutations may have numerous conformational consequences that could lead to improved chromophore formation. Namely: • All three mutations (F99S, M153T, and V163A) found in GFPuv are responsible for precyclized GFPuv forming a tighter-turn than wild-type and S65T-GFPÕs. The S147P mutation also results in a decreased intramolecular distance between the carbonyl carbon of Thr65 and the amide nitrogen of Gly67 relative to that observed in the precyclized structures obtained for S65T-GFP. • If one can assume that an increased Arg96–Tyr66 interaction, which is reflected in a decreased Arg96– Tyr66 distance, can lead to more efficient chromophore formation, then our calculations suggest that the M153T and V163A mutations in GFPuv are partially responsible for the increased maturation efficiency observed in GFPuv by improving the Arg96– Tyr66 interactions. The same is true for the S147P mutation in S147P-GFP. Although the mutations in GFPuv and S147P may improve the thermosensitivity of the thermostable
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GFP mutants for the reasons suggested above, our calculations do not rule out the possibility that other reasons, such as the effects of the mutations on chromophore oxidation, might be responsible for the observed thermostability.
Acknowledgements M.Z. is a Henry Dreyfus Teacher-Scholar. The NIH and Research Corporation provided support for this research. This project was supported in part by NSF Grant CHE-0116435 as part of the MERCURY supercomputer consortium (http://mars.hamilton.edu).
References [1] M. Zimmer, Chem. Rev. 102 (2002) 759. [2] R.Y. Tsien, Annu. Rev. Biochem. 67 (1998) 509. [3] M. Zimmer, Glowing Genes: A Revolution in Biotechnology, Prometheus Books, Amherst, NY, 2005. [4] A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, R.Y. Tsien, TIBS 20 (1995) 448. [5] R. Heim, D.C. Prasher, R.Y. Tsien, Proc. Natl. Acad. Sci. USA 91 (1994) 12501. [6] B.G. Reid, G.C. Flynn, Biochemistry 36 (1997) 6786. [7] M.A. Rosenow, H.A. Huffman, M.E. Phail, R.M. Wachter, Biochemistry 43 (2004) 4464. [8] D.P. Barondeau, C.D. Putnam, C.J. Kassmann, J.A. Tainer, E.D. Getzoff, Proc. Natl. Acad. Sci. USA 100 (2003) 12111. [9] A. Crameri, E.A. Whitehorn, E. Tate, W.P.C. Stemmer, Nat. Biotechnol. 14 (1996) 315. [10] H. Fukuda, M. Arai, K. Kuwajima, Biochemistry 39 (2000) 12025. [11] G.H. Patterson, S.M. Knobel, W.D. Sharif, S.R. Kain, D.W. Piston, Biophys. J. 73 (1997) 2782. [12] Y. Kimata, M. Iwaki, C.R. Lim, K. Kohno, Biochem. Biophys. Res. Commun. 232 (1997) 69.
31
[13] C.R. Lim, Y. Kimata, M. Oka, K. Nomaguchi, K. Kohno, J. Biochem. 118 (1995) 13. [14] R. Battistutta, A. Negro, G. Zanotti, Proteins 41 (2000) 429. [15] M.A. Elsliger, R.M. Wachter, G.T. Hanson, K. Kallio, S.J. Remington, Biochemistry 38 (1999) 5296. [16] F. Mohamadi, N. Richards, W. Guida, R. Liskamp, M. Lipton, C. Caulfield, G. Chang, T. Hendrickson, W. Still, J. Comput. Chem. 11 (1990) 440. [17] W. Still, A. Tempczyk, R. Hawley, T. Hendrickson, J. Am. Chem. Soc. 112 (1990) 6127. [18] G.M. Keseru, I. Kolossvary, J. Am. Chem. Soc. 123 (2001) 12708. [19] I. Kolossvary, G.M. Keseru, J. Comput. Chem. 22 (2001) 21. [20] F. Guarnieri, W. Still, J. Comput. Chem. 15 (1994) 1302. [21] H. Senderowitz, F. Guarnieri, W. Still, J. Am. Chem. Soc. 117 (1995) 8211. [22] P. Shenkin, Q. McDonald, J. Comput. Chem. 15 (1994) 899. [23] W. Kabsch, Acta Crystallogr., Sect. A 32 (1976) 922. [24] W. Kabsch, Acta Crystallogr., Sect. A 34 (1978) 827. [25] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, Nucleic Acids Res. 28 (2000) 235. [26] V. Helms, T.P. Straatsma, J.A. McCammon, J. Phys. Chem. B 103 (1999) 3263. [27] B.R. Branchini, A.R. Nemser, M. Zimmer, J. Am. Chem. Soc. 120 (1998) 1. [28] M. Donnelly, F. Fedeles, M. Wirstam, P.E.M. Siegbahn, M. Zimmer, J. Am. Chem. Soc. 123 (2001) 4679. [29] P.E.M. Siegbahn, M. Wirstam, M. Zimmer, Int. J. Quantum Chem. 81 (2001) 169. [30] M. Zimmer, B.R. Branchini, J.O. Lusins, in: J.W. Hastings, L.J. Kricka, P.E. Stanley (Eds.), Bioluminescence and Chemiluminescence: Proceedings of the 9th International Symposium 1996, Wiley, Chichester, 1996, p. 407. [31] M. Ormoe, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, Science 273 (1996) 1392. [32] F. Yang, L. Moss, G. Phillips, Nat. Biotechnol. 14 (1996) 1246. [33] M.V. Matz, A.F. Fradkov, Y.A. Labas, A.P. Savitisky, A.G. Zaraisky, M.L. Markelov, S.A. Lukyanov, Nat. Biotechnol. 17 (1999) 969. [34] T. Ehrig, D.J. OÕKane, F.G. Pendergast, FEBS Lett. 367 (1995) 163. [35] J. Wiehler, G. Jung, C. Seebacher, Z. Zumbusch, B. Steipe, Chembiochem 4 (2003) 1164.