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The mechanism of dehydration in chromophore maturation of wild-type green fluorescent protein: A theoretical study
Accepted Manuscript Title: The Mechanism of Dehydration in Chromophore Maturation of Wild-type Green Fluorescent Protein: A Theoretical Study Author: Yingying Ma Jian-Guo Yu Qiao Sun Zhen Li Sean C. Smith PII: DOI: Reference:
S0009-2614(15)00316-4 http://dx.doi.org/doi:10.1016/j.cplett.2015.04.061 CPLETT 32970
To appear in: Received date: Revised date: Accepted date:
26-3-2015 28-4-2015 30-4-2015
Please cite this article as:http://dx.doi.org/10.1016/j.cplett.2015.04.061 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical Abstract
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Highlights: 1. The dehydration in chromophore maturation of wild-type GFP is exothermic. 2. The proton of Arg96 transferring to Cβ anion of Tyr66 is the rate-limiting step.
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3. The dehydration/hydration reaction in chromophore maturation of GFP is reversible.
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The Mechanism of Dehydration in Chromophore Maturation of Wild-type Green Fluorescent Protein: A Theoretical Study
Inner Mongolia University of Technology
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Yingying Maa*, Jian-Guo Yub*, Qiao Sunc*, Zhen Lic, Sean C. Smithd
b
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Hohhot 010051, P. R. China
Key Laboratory of Theoretical and Computational Photochemistry
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Ministry of Education,
College of Chemistry, Beijing Normal University,
School of Radiation Medicine and Radiation Protection,
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c
d
Beijing 100875, P. R. China.
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Soochow University,
Suzhou 215123, P.R. China
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Integrated Materials Design Centre, School of Chemical Engineering,
The University of New South Wales, NSW2052, Sydney, Australia.
*Corresponding authors. Email: [email protected]; [email protected];
[email protected] 3
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Abstract An interesting aspect of the green fluorescent protein (GFP) is its autocatalytic chromophore maturation. Numerous experimental studies have indicated that dehydration is the last step in the chromophore maturation process of wild-type GFP. Based on the crystal structure of
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wild-type GFP, the mechanism of the reverse reaction of dehydration was investigated by using density functional theory (DFT) in this study. Our results proposed that the dehydration is
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exothermic. Moreover, the rate-limiting step of the mechanism is the proton on guanidinium of Arg96 transferring to the β-carbon anion of Tyr66, which is consistent with the experimental
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observation.
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Keywords: Density functional theory, Guanidinium of Arg96, Autocatalytic, β-carbon anion of
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Tyr66, Reverse reaction.
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1. Introduction The Green fluorescent protein (GFP) has become an invaluable tool in the past decades for biological sciences research.1-4 The GFP has a barrel-like structure with the chromophore in the
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center. The wild-type GFP exhibits a large absorption peak at 395 nm and a smaller peak at 475 nm, owing to the neutral and anionic chromophores respectively.1,5 An excited state proton
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transfer process occurs upon irradiation of the neutral chromophore, generating the anionic chromophore. The mechanisms of proton transfer in the GFP, as well as structural and
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mechanistic aspects of other fluorescent proteins, have been studied by numerous groups.6-20 The extraordinary utility of GFPs is due to their high quantum yield and relative
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photostability,21 which are due to the chromophores. The formation of chromophore is an autocatalytic process, which is finished by its own three amino acids (Ser65, Tyr66 and Gly67
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for wild-type GFP), and does not need a cofactor.22
In spite of the research described above, the detailed mechanism for the chromophore maturation in GFP is still unclear. There have been some early efforts to investigate this
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mechanism based on experimental observations1,23,24 and theoretical analysis25 respectively.
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In 1994, Tsien’s group1,23 suggested that the first step in chromophore maturation is cyclization,
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followed by dehydration and then a final oxidation step based on experiments. In 2001, based on density functional theory (DFT) studies, Siegbahn et al.25 suggested that oxidized mechanism (dehydrogenation of residue Tyr66 prior to cyclization) is more favorable in energy than the normally accepted reduced mechanism (cyclization precedes dehydrogenation). However, due to potential artifacts associated with the cluster models used, their calculations has been proposed to be problematic.22 In recent years, the mechanism of chromophore maturation of GFP has continued to draw attention. Based on experiments, two mechanisms have been suggested (Figure 1). One mechanism
proposed
by
Getzoff
and
her
coworkers26-31
was
known
as
cyclization-dehydration-oxidation, the other mechanism suggested by Wachter et al.32-37 is cyclization-oxidation-dehydration. In both mechanisms, cyclization is the first process in chromophore maturation of wild-type GFP. The detailed mechanism of cyclization has been investigated.38,39 5
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The above two maturation mechanisms probably occur simultaneously, and the relative flux through each depends on oxygen concentration and efficiency of ring dehydration for the specific fluorescent protein variant. However, for wild-type GFP, it seems that there is more evidence to support the cyclization-oxidation-dehydration mechanism at least in aerobic
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conditions.40 Wachter and coworkers proved that the oxidation step occurs before the dehydration step in the maturation of chromophore in GFP. Keenan et al.41 suggested that after
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a first oxidation process, a final oxidation to form the acylimine of the red chromophore competes with a dehydration to form the green chromophore in kinetics, which showed that the
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last step of the GFP chromophore maturation is dehydration.
Thus the last step in the mechanism of chromophore maturation of wild-type GFP is
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dehydration yielding the green fluorescent chromophore, and there is a reversible dehydration-hydration reaction in this step. Experimental study, for example, X-ray containing
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a mixture of dehydrated and hydrated forms of the fived-membered heterocyclic ring (Y66S avGFP variant) substantially support this dehydration-hydration equilibrium.30 In addition, the crystallographic
data
from
Y66L
avGFP
variant
are
also
consistent
with
a
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dehydration-hydration equilibrium.32,34
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A possible mechanism for the dehydration in chromophore maturation of wild-type GFP
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suggested by Wachter et al. is that the loss of a proton on Cβ66 is the first step and the second step is the elimination of the hydroxyl on five-membered heterocyclic.36 Arg96 was proposed to play a role of base, abstracting the Cβ66 proton yielding carbon anion, which facilitates the elimination of the hydroxyl on the five-membered heterocyclic, a process that is probably facilitated by the carboxylic acid of Glu222 as a proton donor. In addition, they also suggested that the cleavage of C-H bond at the Tyr66 β-carbon is the rate-limiting step of the final process leading to green fluorescence.
However, the detailed reaction mechanism of dehydration in chromophore maturation of wild-type GFP is not known yet. The crystal structure of wild-type of GFP with matured chromophore been solved experimentally42 and since the last step in the maturation of wild-type GFP is dehydration it should be feasible using this crystal structure as a starting point to computationally study the mechanism of dehydration in reverse. This should allow insight to be gained regarding the dehydration mechanism. 6
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In this paper, we will investigate the mechanism of reverse reaction of dehydration based on the crystal structure of wild-type of GFP using DFT method. Our computational results indicate that the first step involves a hydroxyl anion originating from crystallographic water molecule (W24) attacking the five-membered heterocyclic of the chromophore and the second
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step is the proton on guanidinium of Arg96 transferring to the β-carbon anion of Tyr66 (see Figure 2). This study will provide useful information to aid a better understanding of the
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mechanism of the chromophore maturation of GFP. 2. Computational methods
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An active-site model was constructed according to the mechanism described above on the basis of the crystal structure (pdb entry 1GFL42). The truncated model consists of the chromophore,
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the side chain of Arg96, Glu222, Ser205 and His148, the atoms Cα, C and O of Phe64, the crystal water W24 and crystal water 10 (W10). There are 89 atoms in the model whose total
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charge is zero. The initial structure of the reactant was constructed on the basis of crystal structure. All calculations here were carried out using the B3LYP density functional43,44.
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Geometry optimization was performed with the (6-31G(d,p)) basis set. Based on the geometries, more accurate energies were obtained by performing single-point calculations with
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the larger basis set 6-311+G(2d,2p). We also performed frequency calculations to confirm the
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local minimum and transition states. Intrinsic reaction coordinate (IRC)45,46 analyses were performed to confirm that the transition state connect the corresponding two local minimum. Some atoms were frozen to their crystallographic positions during the geometry optimizations to ensure the calculated structures close to those generated experimentally. The frozen atoms are marked with asterisks in the figure 3. Fixing some atoms to their X-ray positions generates a few small negative eigenvalues (only in the order of 10 cm-1) for the optimized structures; however, these do not generate significant effect to the energetic results. The solvation effects from the protein surroundings are calculated at the same theory level as the optimizations by performing single point on top of the optimized geometries using the conductor-like polarizable continuum model (CPCM)47,48. The value of ε=4 was used in modeling protein surroundings. All calculations presented here were performed using Gaussian09 program package49. 7
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3. Results and Discussion 3.1. Nucleophilic attack (Hydroxyl anion attacking the five-membered heterocyclic) The first step of reverse reaction of the dehydration is hydroxyl anion originating from crystallographic W24 attacking the five-membered heterocyclic, and the product of this step is
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the five-membered heterocyclic with a hydroxyl. The optimized geometries of reactant, transition state, and product of this step are listed in Figure 3. It is shown in Figure 3 that the
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distance between OW of W24 and C1 of the five-membered heterocyclic in Re is 3.03 Å, and in its crystal structure, the distance is 3.07 Å, which are consistent with experimental data42. The
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distance between HW of W24 and Oγ of Ser65 is 1.80 Å, and the distance between Hγ of Ser65 and Oε1 of Glu222 carboxylate is 1.61 Å. These distances show that there are hydrogen bond
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interactions between OW of W24 and Oγ of Ser65, between Oγ of Ser65 and Oε1 of Glu222 carboxylate respectively. The hydroxyl anion can be drawn by deprotonation of W24 by
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Glu222 carboxylate via this hydrogen bonding network.
For the transition state of hydroxyl anion attacking five-membered heterocyclic TS1, the
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C1-OW, HW-Oγ, Hγ-Oε1 distances are 2.01, 1.11 and 1.06 Å, respectively. In the nucleophilic attack product Int, the three bond distances are 1.48, 0.98 and 1.00 Å, respectively. These bond
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distances show that accompanying the proton transfer from the W24 to Glu222 carboxylate,
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the hydroxyl anion transfers to the C1 atom of the five-membered heterocyclic. In detail, the nucleophilic transition state TS1 is identified as the first-order saddle point with only one imaginary frequency (340i cm-1), corresponding to C1-OW, HW-Oγ and Hγ-Oε1 simultaneous stretch mode. The mode is strongly bound to the reaction coordinate, which is supported by IRC analyses. The energy of TS1 relative to the reactant Re is 20.8 kcal/mol. Upon addition of surrounding solvation in the form of CPCM, the barrier increases to 23.0 kcal/mol. The intermediate Int is calculated to be 17.8 kcal/mol higher than Re (16.7 kcal/mol including solvation) (Figure 4). The barrier is increased by 2.2 kcal/mol, most likely because the charges of Glu222 carboxylate are quenched in the nucleophilic transition state TS1. The energy of Int is lowered slightly by 1.1 kcal/mol, related to the fact that this step results in the Cβ anion of Tyr66. The solvation effects do not change the energy of this step significantly, probably because of the generation of charge at the Cβ anion of Tyr66 accompanies the quenching of the charge of the Glu222 carboxylate. The computational results show that the hydroxyl anion 8
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attacking five-membered heterocyclic is endothermic. Accordingly, the dissociation of hydroxyl from the five-membered heterocyclic is exothermic.
3.2. Proton transfer (Proton of Arg96 transferring to Cβ anion of Tyr66)
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Here we will discuss the second step of the reverse reaction of dehydration which is corresponding to the proton transfer from Arg96 guanidinium to Tyr66 Cβ anion. The
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optimized geometries of proton transfer reactant Int, transition state TS2, and product Pr of this step are listed in Figure 3. From Table 1 the Hη1-O and Hη2-O distance in Int is 1.69 and
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1.89 Å respectively. Thus, there is hydrogen bonding interaction between O and Hη1, Hη2 respectively, moreover, it can be seen that the hydrogen bonding interaction of Hη1-O is much
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stronger than that of Hη2-O. The Hη1-Cβ and Hη2-Cβ distance in Int is 3.56 and 2.92 Å respectively. Thus, it is Hη2 that transfers to Cβ anion. In the transition state of the proton
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transfer, TS2, the distance between Nη2 and Hη2 is 1.30 Å, and the distance between Hη2 and Cβ is 1.44 Å. In proton transfer product Pr, the distances of Nη2-Hη2 and Hη2-Cβ are 2.22 and 1.10
d
Å respectively. Thus, it can be seen that the proton has transferred from Nη2 to Cβ from Int to Pr. Meanwhile, the Hη2-O, Hη1-O distances are 2.96 and 1.74 Å respectively in TS2 and are 2.79
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and 1.99 Å respectively in Pr. This is shown that when Hη2 transfers to Cβ anion from Int to Pr,
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the hydrogen bond interaction between Hη2 and O is broken, however, the stronger hydrogen bond interaction between Hη1 and O is still kept in this process. The transition state of proton transfer, TS2, is confirmed to be the first-order saddle point with the only one the imaginary frequency (1304i cm-1), which corresponds to Nη2-Hη2 stretch mode. The mode is strongly bound to the reaction coordinate, which is supported by IRC analyses. The energy of TS2 relative to reactant Re is 21.2 kcal/mol in the gas phase (27.9 kcal/mol with solvation included). The energy of Pr was calculated to be 11.8 kcal/mol relative to reactant Re (15.9 kcal/mol with solvation included). In this proton transfer step, the solvation effects are more pronounced than in the case of the nucleophilic attack step. The barrier and the energy of product are increased by 6.7 kcal/mol and 4.1 kcal/mol respectively. It is easy to rationalize the results considering that when the proton of Arg96 guanidinium transfers to the Cβ anion of Tyr66, the charges of Arg96 guanidinium and Tyr66 Cβ anion are quenched. Hence, the computed energies are readily rationalized and would appear to be feasible in vivo. The Proton of Arg96 transferring 9
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to Cβ anion of Tyr66 is endothermic. Accordingly, the cleavage of C-H bond at the Tyr66 β-carbon anion is exothermic. In summary, our computational results show that the rate-limiting step of the whole reverse reaction of the dehydration is the proton transfer step from Arg96 guanidinium to Tyr66 Cβ
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anion, which is consistent with the experimental observation.36 These data are consistent with experimental observation that the dehydrated moiety is more stable, the hydroxide-bond
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species has a low population, and that the dehydration/hydration reaction is reversible 32.
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4. Conclusion
In this paper, the DFT calculations were performed to investigate the reverse reaction
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mechanism of the dehydration constituting the final step in the process of chromophore maturation of wide-type GFP based on the crystal structure (PDB entry 1GFL). There are two
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steps in the reaction: the first step is hydroxyl anion attacking five-membered heterocyclic of chromospheres with the product of five-membered heterocyclic with hydroxyl on it, and the
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second step is the proton of Arg96 guanidinium transferring to Tyr66 Cβ anion. The calculations show that simultaneously with the proton of crystallographic W24 transferring to
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Glu222 carboxylate anion, the hydroxyl anion attacks the five-membered heterocyclic of
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chromophore. The proton transfer from Arg96 guanidinium to Tyr66 Cβ anion is the rate-limiting step in the reverse reaction of dehydration. In addition, our results show that the dehydrated moiety is more stable and has a much higher population than hydroxide-bound species, which are consistent with experimental observations.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 21303009), and science research project of Inner Mongolia University of Technology (ZD201420), and network center of Inner Mongolia University of Technology.
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(30) Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. Biochemistry 2005, 44, 1960. (31) Barondeau, D. P.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. J. Am. Chem. Soc. 2007, 129, 3118. (32) Wachter, R. M. Acc. Chem. Res. 2007, 40, 120. (33) Zhang, L.; Patel, H. N.; Lappe, J. W.; Wachter, R. M. J. Am. Chem. Soc. 2006, 128, 4766. (34) Rosenow, M. A.; Huffman, H. A.; Phail, M. E.; Wachter, R. M. Biochemistry 2004, 43, 4464. (35) Sniegowski, J. A.; Lappe, J. W.; Patel, H. N.; Huffman, H. A.; Wachter, R. M. J. Biol. Chem. 2005, 280,
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(43) Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
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G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
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Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; ; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene,
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M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.02; Gaussian, Inc., Wallingford, CT, 2009.
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Figure captions Figure 1. Two mechanisms of chromophore maturation of wild-type GFP proposed by Getzoff et al. and Wachter et al. respectively.
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Figure 2. The reverse reaction of dehydration in chromophore maturation of wild-type GFP.
Figure 3. The optimized reactant, transition states, intermediate and product in the reverse
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reaction of dehydration in chromophore maturation of wild-type GFP at the B3LYP/6-31G(d,p) level.
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Figure 4. The potential energy profiles of the reverse reaction of dehydration in chromophore maturation of wide-type GFP. Digits in black are the energies of the B3LYP/6-311+G (2d,2p)
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d
M
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level with zero-point correction; digits in red are the energies of cpcm (ε=4).
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Page 13 of 18
O
ip t
Gly67
Tyr66 N
H O O
HN
HO HO
cr
NH
Ser65
N HN
OH
HO -H2O -H+
O2 H2O2
O H
N
OH
HO
HO
-H2O -H+
O
M
H2O 2
H
N
N N
O2
O
H
an
H
us
O
N
N
Ac ce p
te
d
HO
Figure 1
14
Page 14 of 18
ip t cr us an M Ac ce p
te
d
Figure 2
15
Page 15 of 18
ip t
*
* Arg96
cr
Arg96
His148
*
*
3.03 W24 1.80
*
His148 2.01 W24
*
1.11 1.06
W10
1.61
Ser205
*
Glu222
*
*
1.69 1.89 2.92
1.99
1.44
1.48
W10
*
Glu222
W10
Int
2.22 1.10
His148 *
W10
*
Ser205
Glu222
*
*
* *
*
Ac ce p
Ser205
*
His148
te
His148
Arg96
1.30
d
1.74
1.00
*
Arg96
Arg96
0.98
M
TS1
*
*
*
*
Re
*
W10
Ser205
an
Glu222
*
us
*
*
*
TS2
Ser205
*
Glu222
*
Pr
Figure 3
16
Page 16 of 18
ip t cr us
27.9 23.0 17.8
an
20.8
21.2
15.9
16.7
M TS1
Int
TS2
Pr
Ac ce p
te
d
0.0 0.0 Re
11.8
Figure 4
17
Page 17 of 18
Table 1. Important geometry parameters (in Å) in reaction path calculations with B3LYP/6-31G(d,p) method. 1-Re
1-TS1
1-Int
1-TS2
1-Pr
Hη1(Arg96)…O(Tyr66)
1.76
1.70
1.69
1.74
1.99
Hη2(Arg96)…O(Tyr66)
1.79
1.82
1.89
2.96
2.79
Hη1(Arg96)…Cβ(Tyr66)
3.89
3.48
3.56
3.03
3.23
Nη2(Arg96)…Hη2(Arg96)
1.03
1.03
Hη2(Arg96)…Cβ(Tyr66)
3.34
3.36
C1(CRO65)…Ow(W24)
3.03
2.01
Oγ(Ser65)…Hw(W24)
1.80
Hγ(Ser65)…Oε1(Glu222)
1.61
cr
ip t
Distance(Å)
1.30
2.22
2.92
1.44
1.10
1.48
1.44
1.43
1.11
0.98
0.98
0.98
1.06
1.00
1.00
0.99
Ac ce p
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M
an
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1.03
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Page 18 of 18
S0009-2614(15)00316-4 http://dx.doi.org/doi:10.1016/j.cplett.2015.04.061 CPLETT 32970
To appear in: Received date: Revised date: Accepted date:
26-3-2015 28-4-2015 30-4-2015
Please cite this article as:
Ac ce p
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an
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Graphical Abstract
1
Page 1 of 18
Highlights: 1. The dehydration in chromophore maturation of wild-type GFP is exothermic. 2. The proton of Arg96 transferring to Cβ anion of Tyr66 is the rate-limiting step.
Ac ce p
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3. The dehydration/hydration reaction in chromophore maturation of GFP is reversible.
2
Page 2 of 18
The Mechanism of Dehydration in Chromophore Maturation of Wild-type Green Fluorescent Protein: A Theoretical Study
Inner Mongolia University of Technology
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a
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Yingying Maa*, Jian-Guo Yub*, Qiao Sunc*, Zhen Lic, Sean C. Smithd
b
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Hohhot 010051, P. R. China
Key Laboratory of Theoretical and Computational Photochemistry
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Ministry of Education,
College of Chemistry, Beijing Normal University,
School of Radiation Medicine and Radiation Protection,
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c
d
Beijing 100875, P. R. China.
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Soochow University,
Suzhou 215123, P.R. China
d
Integrated Materials Design Centre, School of Chemical Engineering,
The University of New South Wales, NSW2052, Sydney, Australia.
*Corresponding authors. Email: [email protected]; [email protected];
[email protected] 3
Page 3 of 18
Abstract An interesting aspect of the green fluorescent protein (GFP) is its autocatalytic chromophore maturation. Numerous experimental studies have indicated that dehydration is the last step in the chromophore maturation process of wild-type GFP. Based on the crystal structure of
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wild-type GFP, the mechanism of the reverse reaction of dehydration was investigated by using density functional theory (DFT) in this study. Our results proposed that the dehydration is
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exothermic. Moreover, the rate-limiting step of the mechanism is the proton on guanidinium of Arg96 transferring to the β-carbon anion of Tyr66, which is consistent with the experimental
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observation.
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Keywords: Density functional theory, Guanidinium of Arg96, Autocatalytic, β-carbon anion of
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Tyr66, Reverse reaction.
4
Page 4 of 18
1. Introduction The Green fluorescent protein (GFP) has become an invaluable tool in the past decades for biological sciences research.1-4 The GFP has a barrel-like structure with the chromophore in the
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center. The wild-type GFP exhibits a large absorption peak at 395 nm and a smaller peak at 475 nm, owing to the neutral and anionic chromophores respectively.1,5 An excited state proton
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transfer process occurs upon irradiation of the neutral chromophore, generating the anionic chromophore. The mechanisms of proton transfer in the GFP, as well as structural and
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mechanistic aspects of other fluorescent proteins, have been studied by numerous groups.6-20 The extraordinary utility of GFPs is due to their high quantum yield and relative
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photostability,21 which are due to the chromophores. The formation of chromophore is an autocatalytic process, which is finished by its own three amino acids (Ser65, Tyr66 and Gly67
M
for wild-type GFP), and does not need a cofactor.22
In spite of the research described above, the detailed mechanism for the chromophore maturation in GFP is still unclear. There have been some early efforts to investigate this
d
mechanism based on experimental observations1,23,24 and theoretical analysis25 respectively.
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In 1994, Tsien’s group1,23 suggested that the first step in chromophore maturation is cyclization,
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followed by dehydration and then a final oxidation step based on experiments. In 2001, based on density functional theory (DFT) studies, Siegbahn et al.25 suggested that oxidized mechanism (dehydrogenation of residue Tyr66 prior to cyclization) is more favorable in energy than the normally accepted reduced mechanism (cyclization precedes dehydrogenation). However, due to potential artifacts associated with the cluster models used, their calculations has been proposed to be problematic.22 In recent years, the mechanism of chromophore maturation of GFP has continued to draw attention. Based on experiments, two mechanisms have been suggested (Figure 1). One mechanism
proposed
by
Getzoff
and
her
coworkers26-31
was
known
as
cyclization-dehydration-oxidation, the other mechanism suggested by Wachter et al.32-37 is cyclization-oxidation-dehydration. In both mechanisms, cyclization is the first process in chromophore maturation of wild-type GFP. The detailed mechanism of cyclization has been investigated.38,39 5
Page 5 of 18
The above two maturation mechanisms probably occur simultaneously, and the relative flux through each depends on oxygen concentration and efficiency of ring dehydration for the specific fluorescent protein variant. However, for wild-type GFP, it seems that there is more evidence to support the cyclization-oxidation-dehydration mechanism at least in aerobic
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conditions.40 Wachter and coworkers proved that the oxidation step occurs before the dehydration step in the maturation of chromophore in GFP. Keenan et al.41 suggested that after
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a first oxidation process, a final oxidation to form the acylimine of the red chromophore competes with a dehydration to form the green chromophore in kinetics, which showed that the
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last step of the GFP chromophore maturation is dehydration.
Thus the last step in the mechanism of chromophore maturation of wild-type GFP is
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dehydration yielding the green fluorescent chromophore, and there is a reversible dehydration-hydration reaction in this step. Experimental study, for example, X-ray containing
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a mixture of dehydrated and hydrated forms of the fived-membered heterocyclic ring (Y66S avGFP variant) substantially support this dehydration-hydration equilibrium.30 In addition, the crystallographic
data
from
Y66L
avGFP
variant
are
also
consistent
with
a
d
dehydration-hydration equilibrium.32,34
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A possible mechanism for the dehydration in chromophore maturation of wild-type GFP
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suggested by Wachter et al. is that the loss of a proton on Cβ66 is the first step and the second step is the elimination of the hydroxyl on five-membered heterocyclic.36 Arg96 was proposed to play a role of base, abstracting the Cβ66 proton yielding carbon anion, which facilitates the elimination of the hydroxyl on the five-membered heterocyclic, a process that is probably facilitated by the carboxylic acid of Glu222 as a proton donor. In addition, they also suggested that the cleavage of C-H bond at the Tyr66 β-carbon is the rate-limiting step of the final process leading to green fluorescence.
However, the detailed reaction mechanism of dehydration in chromophore maturation of wild-type GFP is not known yet. The crystal structure of wild-type of GFP with matured chromophore been solved experimentally42 and since the last step in the maturation of wild-type GFP is dehydration it should be feasible using this crystal structure as a starting point to computationally study the mechanism of dehydration in reverse. This should allow insight to be gained regarding the dehydration mechanism. 6
Page 6 of 18
In this paper, we will investigate the mechanism of reverse reaction of dehydration based on the crystal structure of wild-type of GFP using DFT method. Our computational results indicate that the first step involves a hydroxyl anion originating from crystallographic water molecule (W24) attacking the five-membered heterocyclic of the chromophore and the second
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step is the proton on guanidinium of Arg96 transferring to the β-carbon anion of Tyr66 (see Figure 2). This study will provide useful information to aid a better understanding of the
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mechanism of the chromophore maturation of GFP. 2. Computational methods
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An active-site model was constructed according to the mechanism described above on the basis of the crystal structure (pdb entry 1GFL42). The truncated model consists of the chromophore,
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the side chain of Arg96, Glu222, Ser205 and His148, the atoms Cα, C and O of Phe64, the crystal water W24 and crystal water 10 (W10). There are 89 atoms in the model whose total
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charge is zero. The initial structure of the reactant was constructed on the basis of crystal structure. All calculations here were carried out using the B3LYP density functional43,44.
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Geometry optimization was performed with the (6-31G(d,p)) basis set. Based on the geometries, more accurate energies were obtained by performing single-point calculations with
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the larger basis set 6-311+G(2d,2p). We also performed frequency calculations to confirm the
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local minimum and transition states. Intrinsic reaction coordinate (IRC)45,46 analyses were performed to confirm that the transition state connect the corresponding two local minimum. Some atoms were frozen to their crystallographic positions during the geometry optimizations to ensure the calculated structures close to those generated experimentally. The frozen atoms are marked with asterisks in the figure 3. Fixing some atoms to their X-ray positions generates a few small negative eigenvalues (only in the order of 10 cm-1) for the optimized structures; however, these do not generate significant effect to the energetic results. The solvation effects from the protein surroundings are calculated at the same theory level as the optimizations by performing single point on top of the optimized geometries using the conductor-like polarizable continuum model (CPCM)47,48. The value of ε=4 was used in modeling protein surroundings. All calculations presented here were performed using Gaussian09 program package49. 7
Page 7 of 18
3. Results and Discussion 3.1. Nucleophilic attack (Hydroxyl anion attacking the five-membered heterocyclic) The first step of reverse reaction of the dehydration is hydroxyl anion originating from crystallographic W24 attacking the five-membered heterocyclic, and the product of this step is
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the five-membered heterocyclic with a hydroxyl. The optimized geometries of reactant, transition state, and product of this step are listed in Figure 3. It is shown in Figure 3 that the
cr
distance between OW of W24 and C1 of the five-membered heterocyclic in Re is 3.03 Å, and in its crystal structure, the distance is 3.07 Å, which are consistent with experimental data42. The
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distance between HW of W24 and Oγ of Ser65 is 1.80 Å, and the distance between Hγ of Ser65 and Oε1 of Glu222 carboxylate is 1.61 Å. These distances show that there are hydrogen bond
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interactions between OW of W24 and Oγ of Ser65, between Oγ of Ser65 and Oε1 of Glu222 carboxylate respectively. The hydroxyl anion can be drawn by deprotonation of W24 by
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Glu222 carboxylate via this hydrogen bonding network.
For the transition state of hydroxyl anion attacking five-membered heterocyclic TS1, the
d
C1-OW, HW-Oγ, Hγ-Oε1 distances are 2.01, 1.11 and 1.06 Å, respectively. In the nucleophilic attack product Int, the three bond distances are 1.48, 0.98 and 1.00 Å, respectively. These bond
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distances show that accompanying the proton transfer from the W24 to Glu222 carboxylate,
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the hydroxyl anion transfers to the C1 atom of the five-membered heterocyclic. In detail, the nucleophilic transition state TS1 is identified as the first-order saddle point with only one imaginary frequency (340i cm-1), corresponding to C1-OW, HW-Oγ and Hγ-Oε1 simultaneous stretch mode. The mode is strongly bound to the reaction coordinate, which is supported by IRC analyses. The energy of TS1 relative to the reactant Re is 20.8 kcal/mol. Upon addition of surrounding solvation in the form of CPCM, the barrier increases to 23.0 kcal/mol. The intermediate Int is calculated to be 17.8 kcal/mol higher than Re (16.7 kcal/mol including solvation) (Figure 4). The barrier is increased by 2.2 kcal/mol, most likely because the charges of Glu222 carboxylate are quenched in the nucleophilic transition state TS1. The energy of Int is lowered slightly by 1.1 kcal/mol, related to the fact that this step results in the Cβ anion of Tyr66. The solvation effects do not change the energy of this step significantly, probably because of the generation of charge at the Cβ anion of Tyr66 accompanies the quenching of the charge of the Glu222 carboxylate. The computational results show that the hydroxyl anion 8
Page 8 of 18
attacking five-membered heterocyclic is endothermic. Accordingly, the dissociation of hydroxyl from the five-membered heterocyclic is exothermic.
3.2. Proton transfer (Proton of Arg96 transferring to Cβ anion of Tyr66)
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Here we will discuss the second step of the reverse reaction of dehydration which is corresponding to the proton transfer from Arg96 guanidinium to Tyr66 Cβ anion. The
cr
optimized geometries of proton transfer reactant Int, transition state TS2, and product Pr of this step are listed in Figure 3. From Table 1 the Hη1-O and Hη2-O distance in Int is 1.69 and
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1.89 Å respectively. Thus, there is hydrogen bonding interaction between O and Hη1, Hη2 respectively, moreover, it can be seen that the hydrogen bonding interaction of Hη1-O is much
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stronger than that of Hη2-O. The Hη1-Cβ and Hη2-Cβ distance in Int is 3.56 and 2.92 Å respectively. Thus, it is Hη2 that transfers to Cβ anion. In the transition state of the proton
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transfer, TS2, the distance between Nη2 and Hη2 is 1.30 Å, and the distance between Hη2 and Cβ is 1.44 Å. In proton transfer product Pr, the distances of Nη2-Hη2 and Hη2-Cβ are 2.22 and 1.10
d
Å respectively. Thus, it can be seen that the proton has transferred from Nη2 to Cβ from Int to Pr. Meanwhile, the Hη2-O, Hη1-O distances are 2.96 and 1.74 Å respectively in TS2 and are 2.79
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and 1.99 Å respectively in Pr. This is shown that when Hη2 transfers to Cβ anion from Int to Pr,
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the hydrogen bond interaction between Hη2 and O is broken, however, the stronger hydrogen bond interaction between Hη1 and O is still kept in this process. The transition state of proton transfer, TS2, is confirmed to be the first-order saddle point with the only one the imaginary frequency (1304i cm-1), which corresponds to Nη2-Hη2 stretch mode. The mode is strongly bound to the reaction coordinate, which is supported by IRC analyses. The energy of TS2 relative to reactant Re is 21.2 kcal/mol in the gas phase (27.9 kcal/mol with solvation included). The energy of Pr was calculated to be 11.8 kcal/mol relative to reactant Re (15.9 kcal/mol with solvation included). In this proton transfer step, the solvation effects are more pronounced than in the case of the nucleophilic attack step. The barrier and the energy of product are increased by 6.7 kcal/mol and 4.1 kcal/mol respectively. It is easy to rationalize the results considering that when the proton of Arg96 guanidinium transfers to the Cβ anion of Tyr66, the charges of Arg96 guanidinium and Tyr66 Cβ anion are quenched. Hence, the computed energies are readily rationalized and would appear to be feasible in vivo. The Proton of Arg96 transferring 9
Page 9 of 18
to Cβ anion of Tyr66 is endothermic. Accordingly, the cleavage of C-H bond at the Tyr66 β-carbon anion is exothermic. In summary, our computational results show that the rate-limiting step of the whole reverse reaction of the dehydration is the proton transfer step from Arg96 guanidinium to Tyr66 Cβ
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anion, which is consistent with the experimental observation.36 These data are consistent with experimental observation that the dehydrated moiety is more stable, the hydroxide-bond
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species has a low population, and that the dehydration/hydration reaction is reversible 32.
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4. Conclusion
In this paper, the DFT calculations were performed to investigate the reverse reaction
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mechanism of the dehydration constituting the final step in the process of chromophore maturation of wide-type GFP based on the crystal structure (PDB entry 1GFL). There are two
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steps in the reaction: the first step is hydroxyl anion attacking five-membered heterocyclic of chromospheres with the product of five-membered heterocyclic with hydroxyl on it, and the
d
second step is the proton of Arg96 guanidinium transferring to Tyr66 Cβ anion. The calculations show that simultaneously with the proton of crystallographic W24 transferring to
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Glu222 carboxylate anion, the hydroxyl anion attacks the five-membered heterocyclic of
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chromophore. The proton transfer from Arg96 guanidinium to Tyr66 Cβ anion is the rate-limiting step in the reverse reaction of dehydration. In addition, our results show that the dehydrated moiety is more stable and has a much higher population than hydroxide-bound species, which are consistent with experimental observations.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 21303009), and science research project of Inner Mongolia University of Technology (ZD201420), and network center of Inner Mongolia University of Technology.
10
Page 10 of 18
References and Notes (1) Heim, R.; Prasher, D. C.; Tsien, R. Y. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 12501. (2) Orm, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J. Science 1996, 273, 1392. (3) Zimmer, M. Chem. Rev. 2002, 102, 759.
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(4) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217.
(5) Niwa, H.; Inouye, S.; Hirano, T.; Matsuno, T.; Kojima, S.; Kubota, M.; Ohashi, M.; Tsuji, F. l. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13617.
cr
(6) Wang, S. F.; Smith, S. C. Phys. Chem. Chem. Phys. 2007, 9, 452.
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(9) Vendrell, O.; Gelabert, R.; Moreno, M.; Lluch, J. M. J. Phys. Chem. B 2008, 112, 5500.
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(12) Olsen, S.; Prescott, M.; Wilmann, P.; Battad, J.; Rossjohn, J.; Smith, S. C. Chem. Phys. Lett. 2006, 420, 507.
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(14) Schäfer, L. V.; Groenhof, G.; Boggio-Pasqua, M.; Robb, M. A.; Grubmüller, H. PLoS Comput Biol 2008,
M
4, e1000034.
(15) Andresen, M.; Wahl, M. C.; Stiel, A. C.; Grāter, F.; Schāfer, L. V.; Trowitzsch, S.; Weber, G.; Eggeling, C.; Grubmüller, H.; Hell, S. W.; Jakobs, S. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 13070.
d
(16) Sun, Q.; Li, Z.; Lan, Z.; Pfisterer, C.; Doerr, M.; Fischer, S.; Smith, S. C.; Thiel, W. Phys. Chem. Chem. Phys. 2012, 14, 11413.
te
(17) Zhang, H.; Sun, Q.; Wang, S.; Olsen, S.; Smith, S. C. Theoretical Studies of Green and Red Fluorescent Proteins. In Hydrogen Bonding and Transfer in the Excited State; John Wiley & Sons, Ltd: New York, 2011; pp 815.
Ac ce p
(18) Vendrell, O.; Gelabert, R.; Moreno, M.; Lluch, J. M. J. Am. Chem. Soc. 2006, 128, 3564. (19) Schäfer, L. V.; Gerrit, G.; Klingen, A. R.; Ullmann, G. M.; Martial, B.-P.; Robb, M. A.; Helmut, G.
Angew. Chem. Int. Ed. 2007, 46, 530.
(20) Iizuka, R.; Yamagishi-Shirasaki, M.; Funatsu, T. Anal. Biochem. 2011, 414, 173.
(21) Day, R. N.; Davidson, M. W. Chem. Soc. Rev. 2009, 38, 2887. (22) Lemay, N. P.; Morgan, A. L.; Archer, E. J.; Dickson, L. A.; Megley, C. M.; Zimmer, M. Chem. Phys.
2008, 348, 152.
(23) Cubitt, A. B.; Heim, R.; Adams, S. R.; Boyd, A. E.; Gross, L. A.; Tsien, R. Y. Trends Biochem. Sci.
1995, 20, 448.
(24) Reid, B. G.; Flynn, G. C. Biochemistry 1997, 36, 6786. (25) Per, E. M. S.; Maria, W.; Marc, Z. Int. J. Quantum Chem. 2001, 81, 169. (26) Wood, T. I.; Barondeau, D. P.; Hitomi, C.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. Biochemistry 2005, 44, 16211. (27) Barondeau, D. P.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. J. Am. Chem. Soc. 2006, 128, 4685. (28) Barondeau, D. P.; Putnam, C. D.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 12111. (29) Barondeau, D. P.; Tainer, J. A.; Getzoff, E. D. J. Am. Chem. Soc. 2006, 128, 3166. 11
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(30) Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. Biochemistry 2005, 44, 1960. (31) Barondeau, D. P.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. J. Am. Chem. Soc. 2007, 129, 3118. (32) Wachter, R. M. Acc. Chem. Res. 2007, 40, 120. (33) Zhang, L.; Patel, H. N.; Lappe, J. W.; Wachter, R. M. J. Am. Chem. Soc. 2006, 128, 4766. (34) Rosenow, M. A.; Huffman, H. A.; Phail, M. E.; Wachter, R. M. Biochemistry 2004, 43, 4464. (35) Sniegowski, J. A.; Lappe, J. W.; Patel, H. N.; Huffman, H. A.; Wachter, R. M. J. Biol. Chem. 2005, 280,
ip t
26248.
(36) Pouwels, L. J.; Zhang, L.; Chan, N. H.; Dorrestein, P. C.; Wachter, R. M. Biochemistry 2008, 47, 10111. (37) Wachter, R. M. “Mechanistic aspects of GFP chromophore biogenesis”; Genetically Engineered Probes
cr
for Biomedical Applications, 2006, San Jose, CA, USA.
(38) Ma, Y. Y.; Sun, Q.; Zhang, H.; Peng, L.; Yu, J.-G.; Smith, S. C. J. Phys. Chem. B 2010, 114, 9698. (39) Ma, Y. Y.; Sun, Q.; Li, Z.; Yu, J.-G.; Smith, S. C. J. Phys. Chem. B 2012, 116, 1426.
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(40) Craggs, T. D. Chem. Soc. Rev. 2009, 38, 2865.
(41) Strack, R. L.; Strongin, D. E.; Mets, L.; Glick, B. S.; Keenan, R. J. J. Am. Chem. Soc. 2010, 132, 8496. (42) Yang, F.; Moss, L. G.; Phillips, G. N. Nat. Biotechnol. 1996, 14, 1246.
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(43) Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
(44) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785.
(45) Gonzalez, C.; Schlegel, H. B. J. Chem. Phys. 1989, 90, 2154. (46) Gonzalez, C.; Schlegel, H. B. J. Phys. Chem. 1990, 94, 5523.
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(47) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102, 1995.
(48) Cammi, R.; Mennucci, B.; Tomasi, J. J. Phys. Chem. A 1999, 103, 9100. (49) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani,
d
G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
te
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; ; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene,
Ac ce p
M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.02; Gaussian, Inc., Wallingford, CT, 2009.
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Figure captions Figure 1. Two mechanisms of chromophore maturation of wild-type GFP proposed by Getzoff et al. and Wachter et al. respectively.
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Figure 2. The reverse reaction of dehydration in chromophore maturation of wild-type GFP.
Figure 3. The optimized reactant, transition states, intermediate and product in the reverse
cr
reaction of dehydration in chromophore maturation of wild-type GFP at the B3LYP/6-31G(d,p) level.
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Figure 4. The potential energy profiles of the reverse reaction of dehydration in chromophore maturation of wide-type GFP. Digits in black are the energies of the B3LYP/6-311+G (2d,2p)
Ac ce p
te
d
M
an
level with zero-point correction; digits in red are the energies of cpcm (ε=4).
13
Page 13 of 18
O
ip t
Gly67
Tyr66 N
H O O
HN
HO HO
cr
NH
Ser65
N HN
OH
HO -H2O -H+
O2 H2O2
O H
N
OH
HO
HO
-H2O -H+
O
M
H2O 2
H
N
N N
O2
O
H
an
H
us
O
N
N
Ac ce p
te
d
HO
Figure 1
14
Page 14 of 18
ip t cr us an M Ac ce p
te
d
Figure 2
15
Page 15 of 18
ip t
*
* Arg96
cr
Arg96
His148
*
*
3.03 W24 1.80
*
His148 2.01 W24
*
1.11 1.06
W10
1.61
Ser205
*
Glu222
*
*
1.69 1.89 2.92
1.99
1.44
1.48
W10
*
Glu222
W10
Int
2.22 1.10
His148 *
W10
*
Ser205
Glu222
*
*
* *
*
Ac ce p
Ser205
*
His148
te
His148
Arg96
1.30
d
1.74
1.00
*
Arg96
Arg96
0.98
M
TS1
*
*
*
*
Re
*
W10
Ser205
an
Glu222
*
us
*
*
*
TS2
Ser205
*
Glu222
*
Pr
Figure 3
16
Page 16 of 18
ip t cr us
27.9 23.0 17.8
an
20.8
21.2
15.9
16.7
M TS1
Int
TS2
Pr
Ac ce p
te
d
0.0 0.0 Re
11.8
Figure 4
17
Page 17 of 18
Table 1. Important geometry parameters (in Å) in reaction path calculations with B3LYP/6-31G(d,p) method. 1-Re
1-TS1
1-Int
1-TS2
1-Pr
Hη1(Arg96)…O(Tyr66)
1.76
1.70
1.69
1.74
1.99
Hη2(Arg96)…O(Tyr66)
1.79
1.82
1.89
2.96
2.79
Hη1(Arg96)…Cβ(Tyr66)
3.89
3.48
3.56
3.03
3.23
Nη2(Arg96)…Hη2(Arg96)
1.03
1.03
Hη2(Arg96)…Cβ(Tyr66)
3.34
3.36
C1(CRO65)…Ow(W24)
3.03
2.01
Oγ(Ser65)…Hw(W24)
1.80
Hγ(Ser65)…Oε1(Glu222)
1.61
cr
ip t
Distance(Å)
1.30
2.22
2.92
1.44
1.10
1.48
1.44
1.43
1.11
0.98
0.98
0.98
1.06
1.00
1.00
0.99
Ac ce p
te
d
M
an
us
1.03
18
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