New Insights on the Mechanism of Cyclization in Chromophore

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New Insights on the Mechanism of Cyclization in Chromophore Maturation of Wild-Type Green Fluorescence Protein: A Computational Study Yingying Ma, Hao Zhang, Qiao Sun, and Sean C. Smith J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b04406 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on May 30, 2016

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The Journal of Physical Chemistry

New Insights on the Mechanism of Cyclization in Chromophore Maturation of Wild-Type Green Fluorescence Protein: A Computational Study

Yingying Maa,b*, Hao Zhangc, Qiao Sund*, Sean C. Smithe*

a

b

Institue of Mining Technology, Inner Mongolia University of Technology Hohhot 010051, P. R. China

Inner Mongolia Key Laboratory of Theoretical and Computational Chemistry Simulation Hohhot 010051, P. R. China

c

College of Life Science and Engineering, Northwest University for Nationalities, Lanzhou 730030, P. R. China d

School of Radiation Medicine and Radiation Protection, Soochow University, Suzhou 215123, P. R. China e

Integrated Materials Design Centre, School of Chemical Engineering,

The University of New South Wales, NSW2052, Sydney, Australia.

*Corresponding authors. *(Y.M.): Email: [email protected], Phone: +86-471-3602768 *(Q.S.): Email: [email protected], Phone: +86-512-65882931 *(S.C.S.): Email: [email protected], Phone: +612-93855132

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Abstract Cyclization is the first step in the chromophore maturation process of the green fluorescent protein (GFP). In our previous paper [J. Phys. Chem. B 2012, 116, 14261436], the results of molecular dynamics simulation suggested the possibility that the amide nitrogen atom of Gly67 attacks the carbonyl carbon of Ser65 directly to complete the cyclization process (one-step mechanism). In this paper, density functional theory (DFT) and quantum mechanical/molecular mechanical (QM/MM) calculations were undertaken to study this step reaction in detail. Three cluster model systems (model A, model B and model C) and large protein system were set up to investigate the cyclization process. Our results indicate that the one-step mechanism only exists in the two minimum models. However, in model C and the large protein system, the cyclization mechanism involves two steps: the first step is proton of Gly67 amide nitrogen transferring to carbonyl oxygen of Ser65, generating protonated amide, which is stabilized by a hydrogen bond interaction with a crystallographic water molecule, and the second step is Gly67 amide nitrogen attacking the carbonyl carbon of Ser65. Arg96 plays an important role in promoting the cyclization. The energy of cyclized product relative to reactant is about 10.0 kcal/mol endothermic, which is in line with the experimental results.

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1. Introduction The Green fluorescent protein (GFP) has been widely used for biological sciences research.1 The remarkable utility of GFPs is due to their photostability, and their chromophores are developed in an autocatalytic process.2 The autocatalytic cyclization of wild-type GFP is accomplished by its own three amino acids Ser65, Tyr66 and Gly67.3, 4 The mechanism of GFP chromophore maturation has always attracted attention. In 1994, Tsien et al.3, 5 suggested that the first step in the formation of chromophore is Gly67 amide nitrogen (N67) attacking on Ser65 carbonyl carbon (C65), followed by dehydration and then an oxidation step on the basis of experiment. Siegbahn et al.6 proposed that dehydrogenation of residue Tyr66 prior to cyclization is more favorable in energy than the mechanism that cyclization precedes dehydrogenation based on the density functional calculations. However, their cluster models used do not rely on the trustworthy experiment data.2 Recently, with the development of experimental technology, two mechanisms have been suggested. One mechanism proposed by Getzoff’s group7-12 was called cyclization-dehydration-oxidation, the other mechanism proposed by Wachter et al.1320

is

cyclization-oxidation-dehydration.

The

two

mechanisms

may

occur

simultaneously, however, for wild-type GFP, more evidence seems to support that dehydration is the last step in the maturation mechanism in aerobic conditions. The mechanism of dehydration has been investigated by us.21 Lots of evidence proposed that the first step in chromophore maturation of wild-type GFP is cyclization. Experimental results suggested that cyclization is reversible,8,

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with the cyclized

product being higher in energy. The thermodynamically unfavorable cyclized product is trapped by subsequent reaction, likely ring oxidation under highly aerobic conditions for wild-type GFP.17 Moreover, experimental observation proposed primarily electrostatic role for R96 in chromophore formation, which is to stabilizing the cyclized moiety.10 However, the detailed mechanism of cyclization in chromophore maturation of GFP is still not clear. Two proposed mechanism of cyclization in chromophore maturation of wild-type GFP have been investigated with cluster models firstly,22 which suggest that backbone condensation initiated by deprotonation of the Gly67 amide nitrogen is

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easier than deprotonation of the Tyr66 α-carbon. Then, the effects of protein environment to the above two reaction mechanisms were investigated with molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) method.23 The results show that the cyclization is neither initiated by deprotonation of Gly67 amide nitrogen nor initiated by deprotonation of Tyr66 α-carbon. And the MD results23 show that the distance between amide nitrogen atom of Gly67 and the carbonyl carbon of Ser65 is much less than the sum of their van der Waals radii, indicating that the cyclization process is completed by amide nitrogen atom of Gly67 attacking the carbonyl carbon of Ser65 directly, which means that the proton on Gly67 amide nitrogen transferring to Ser65 carbonyl oxygen, and the Gly67 amide nitrogen attacking the Ser65 carbonyl carbon is a concerted process (Figure 1a). In 2013, Alkorta et al. studied this concerted mechanism of cyclization using a simple model.24 The energy barriers corresponding to the cyclization are 63.5 kcal/mol, but the addition of a water molecule between Gly67 amide nitrogen and Ser65 carbonyl oxygen to assist the proton transfer, dramatically lowers the energy barriers by 21.1 kcal/mol. But in our paper,23 the results of 10 ns MD showed that there is no water between Gly67 amide nitrogen and Ser65 carbonyl oxygen to assist the proton transfer. Moreover, the effects of residues around the chromophore were not considered in their model. Therefore, in this paper we investigated the process of cyclization in detail by using density functional theory (DFT) and QM/MM analysis based on the reliable crystal structure. Our results show that the cyclization is not a concerted process, an intermediate between the precyclized reactant and the cyclized product was found (Figure 1b).

2. Computational methods 2.1 Truncated Active-site Model Three active-site models were constructed on the basis of the crystal structure (pdb entry 2AWJ10): model A, model B and model C. There are only three residues in the model A: Ser65, Tyr66 and Gly67. To simplify calculation, the amino and hydroxyl group of the Ser65, the carboxyl group of the Gly67 are truncated and saturated with H but all atoms of Tyr66 were kept in the model. The model A contains 36 atoms in

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total, and the total charge is 0 (Figure 2a). Compared to model A, Arg96 was added in the model B. To simplify calculation, the amino group and carboxyl group of the Arg96 are truncated and saturated with H. The model B contains 55 atoms in total, and the total charge is +1 (Figure 2b). The model C contains the Ser65, Tyr66, Gly67, Arg96, His148, Ser205, Glu222, crystal water 1042 (W1) and crystal water 1158 (W2). Similarly, some atoms were truncated and saturated with H. Model C contains 90 atoms in total, and the total charge is 0 (Figure 3). All calculations here were performed with the B3LYP25-27 density functional. Geometry optimizations were carried out using the 6-31G(d,p) basis set. Based on the geometries, more accurate energies were obtained by implementing single-point calculations with the larger basis set 6-311+G(2d,2p). Frequency calculations were carried out to confirm the transition state and local minimum. Intrinsic reaction coordinate (IRC28, 29) analyses were performed to identify that the transition state connect the corresponding two local minimums. 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. The conductor-like polarisable continuum model (CPCM30, 31) was applied to calculate the solvation effects from the protein surroundings (ε=4). 2.2 QM/MM Calculation To check the influence of the protein environment on the cyclized mechanism, one snapshot, serving as initial structure for QM/MM optimization, was taken from MD trajectories, which has been published in our last paper.23 The details of QM/MM calculation are exactly the same as the description in our previous paper.23 A twolayer ONIOM32-36 scheme was used for the calculations, and the interface between the QM and MM regions is treated by hydrogen link atoms. The whole ONIOM system contains 12182 atoms in total. The Ser65, Tyr66, Gly67, the side chain of Arg96 and Glu222, the atoms N, H, Cα, Hα, Cβ, C of Val68, the atoms C, O, Cα of Phe64, crystal water W1 and crystal water W2 were treated quantum mechanically. The QM part has 83 atoms in total, and link atoms were attached to Cα of Arg96, Cα of Glu222, Cα of Phe64, Cβ of Val68, and C of Val68. In this study, the QM method was the density functional B3LYP25-27 with a 6-31G(d,p) basis set, and the MM method was the Amber96 force field. Geometries were optimized in the ONIOM-ME (mechanical

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embedding) and ONIOM-EE (electronic embedding) scheme respectively. All calculations presented in this paper were performed using Gaussian 09 program package.37 3. Results and Discussions 3.1 One-step mechanism The one-step mechanism is that when the amide nitrogen atom of Gly67 attacks the carbonyl carbon of Ser65, a proton on N67 (H67) transfers to carbonyl oxygen of Ser65 (O65). In model A, the distance C65-N67 is 3.68 Å in A-Re. In A-TS, the distance C65-N67 and O65-H67 are 2.25 and 0.99 Å respectively. In A-Pr, the two distances are 1.47 and 0.97 Å respectively. The transition state is characterized by an imaginary frequency of 327.2i cm-1, which mainly corresponds to the bond of C65N67 and N67-H67 simultaneous stretch mode. The calculated energy of A-TS is 50.8 kcal/mol (49.6 kcal/mol without solvation) higher than that of A-Re (Figure 4). The energy of A-Pr is calculated to be 13.9 kcal/mol (13.6 kcal/mol without solvation) relative to reactant A-Re. In model B, the distance between the hydrogen on Arg96 guanidinium (Hη) and Tyr66 carbonyl oxygen (O66) is 1.86 Å, and the distance C65-N67 is 3.19 Å in B-Re, which is much shorter than the corresponding value in model A. In B-TS, the distance C65-N67 and O65-H67 are 2.37 and 0.97 Å respectively, and in B-Pr, the two distances are 1.50 and 0.97 Å respectively. The distance Hη-O66 is 1.74 Å in B-TS and 1.86 Å in B-Pr, which show that Arg96 provides stabilization to the transition state of nucleophilic attack. The transition state is characterized by an imaginary frequency of 133.3i cm-1, which mainly corresponds to the bond of C65-N67 stretch and the O65-H67 bond rock out of plane mode. The calculated energy of B-TS is 43.2 kcal/mol (40.2 kcal/mol without solvation) higher than that of B-Re, which is 7.6 kcal/mol lower than the corresponding barrier of model A. The energy of B-Pr was calculated to be 16.1 kcal/mol (17.0 kcal/mol without solvation) relative to reactant BRe (Figure 4). It can be seen that Arg96 plays a role of shortening the distance C65N67, and lowering the barrier of cyclization, which is consistent to the experimental observation that the Arg96 positive charge plays an important role in accelerating the peptide backbone cyclization in GFP.10

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When we tried to enlarge the model, for instance, add Glu222 and keep the hydroxyl of Ser65 in model B, optimize the corresponding concerted nucleophilic attack transition state, we didn’t get stable geometry of the transition state. Then, we tried to optimize the concerted nucleophilic attack transition state in model C and large protein system respectively, but no stable geometry of the transition sate is found. Therefore, we conclude that the one-step mechanism just exists in small models, but does not exist in real protein system. 3.2 Two-step mechanism Two-step mechanism means that there are two steps in the cyclization process of chromophore maturation of wild-type GFP: first, the proton of Gly67 amide nitrogen transfers to carbonyl oxygen of Ser65, and secondly, Gly67 amide nitrogen attacks the carbonyl carbon of Ser65. The model C and the ONIOM system were constructed to illuminate the two-step mechanism. 3.2.1 Proton transfer The first step of cyclization in chromophore maturation is that proton of Gly67 amide nitrogen transfers to carbonyl oxygen of Ser65, generating protonated amide. In model C, the optimized geometry of reactant Re of this step is listed in Figure 3. It can be seen that there are some hydrogen bond networks around precyclized chromophore. For details, three residues have hydrogen bond network with Tyr66: the Hη (Arg96) and O66 has hydrogen bond interaction, and the distance between them is 1.75 Å (Table 1); there is a hydrogen bond between Oη of Tyr66 and Hε of His148, and the distance between them is 1.92 Å; there is a hydrogen bond between Hη of Tyr66 and Oγ of Ser205, and the distance between them is 1.67 Å. Two residues have hydrogen bond network with Ser65: one is hydrogen bond interaction between hydrogen of Ser65 (Hγ) and oxygen of Glu222 carboxylate (Oε1), and corresponding distance is 1.71 Å, the other is a hydrogen bond between O65 and hydrogen of W1, and the distance between them is 2.03 Å. In addition, there is a hydrogen bond between Hγ of Ser205 and Oε1 of Glu222, and the distance between them is 1.62 Å. There are two water molecules between Hα of Tyr66 and oxygen atom of Glu222 carboxylate (Oε2), which means there is a hydrogen bond network between Tyr66 and Glu222. In detail, the distance between Hα of Tyr66 and oxygen atom of W2 (O2) is

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2.08 Å, and the distance between hydrogen of W2 and oxygen atom of W1 is 1.90 Å, and distance between hydrogen of W1 and Oε2 of Glu222 carboxylate is 1.87 Å. All these hydrogen bond interactions have contributions to the chromophore maturation. It is shown in Figure 3 that, the distance O65-H67 is 1.71 Å in Re, which show that there is a hydrogen bond interaction between amide nitrogen of Gly67 and carbonyl oxygen of Ser65. Thus, proton on amide nitrogen of Gly67 can transfer to carbonyl oxygen of Ser65. The distance C65-O65 is 1.25 Å. In TS1, the N67-H67 and O65-H67 distance is 2.25 and 1.00 Å respectively, and C65-O65 distance has been elongated to 1.30 Å. In the proton transfer product Int, the N67-H67, O65-H67 and C65-O65 distances are 3.72, 1.05 and 1.29 Å respectively. These data show that proton on Gly67 amide nitrogen has been transferred to Ser65 carbonyl oxygen. It can be seen that the distance between H67 and O2 is 1.48 Å in Int, which show that there is hydrogen bond interaction between H67 and W2. Thus, our results give the following conclusion: after the proton on Gly67 transferred to carbonyl oxygen of Ser65, generating protonated amide (protonated on the carbonyl oxygen), which is not a stable species, and is stabilized by a hydrogen bonding interaction with the water molecule (W2). In addition, the distance between carbonyl carbon of Tyr66 (C66) and N67 is 1.33 Å in Re to 1.29 Å at TS1 and 1.29 Å at Int, and the distance C66-O66 is 1.26 Å in Re to 1.30 Å at TS1 and 1.30 Å at Int. All these show that the amide of Gly67 has changed into amide anion accompanying the process of proton transfer. We further notice that the distance between Hη of Arg96 and the O66 is 1.75 Å in Re to 1.60 Å in TS1 and 1.54 Å in Int, which show that the Arg96 provides electrostatic stabilization to the intermediate Int, and this is in line with experimental observation that Arg96 has a functional role in formation of the cyclized moiety.10 The proton transfer transition state TS1 is identified as the first-order saddle point with only one imaginary frequency (287.7i cm-1), which corresponds to the O-H bond rock out of plane 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 30.9 kcal/mol (23.4 kcal/mol without solvation). The intermediate Int is calculated to be 27.6 kcal/mol (19.4 kcal/mol without solvation) higher than Re (Figure 5). It can be seen that the barrier and the energy of intermediate are increased by 7.5 and 8.2

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kcal/mol respectively upon addition of solvation, most likely because generation of positive charges at the carbonyl of Ser65 and negative charges at carbonyl of Tyr66. In ONIOM-ME case, the optimized geometries of reactant, transition state and product of this step are listed in Figure 6a. It is shown in Figure 6a that in Re, the distance N67-H67, O65-H67 and C65-O65 are 1.02, 1.95 and 1.24 Å, respectively (Table 2). In TS1, the three distances are 2.30, 0.98 and 1.30 Å respectively. In the proton transfer product Int, the three distances are 3.75, 1.05 and 1.28 Å respectively. The distance between the proton H67 and oxygen of W1 is 1.47 Å. These data show that proton on Gly67 amide nitrogen has been transferred to Ser65 carbonyl oxygen generating protonated amide, which is stabilized by a hydrogen interaction with the water molecule W1. In particular, the distance between Hη (Arg96) and the O66 is 1.67 Å in Re to 1.64 Å in TS1 and 1.65 Å in Int, which show that Arg96 plays a role of stabilizing the proton transfer transition state and intermediate Int. The energy of TS1 relative to the reactant Re is 29.4 kcal/mol, which is similar to the result of model C (30.9 kcal/mol). The intermediate Int is calculated to be 21.0 kcal/mol higher than Re (Figure 7). From comparing the geometries of model C with the geometries in ONIOM-ME case, it can be seen that: first, the distance between Arg96 guanidinium and Tyr66 carbonyl in model C is much smaller than the corresponding distance in ONIOM-ME case. The reason is that in model C Arg96 is truncated at its Cα position, and it can rotate, so that the plane determined by Arg96 guanidinium is almost parallel to the plane determined by Tyr66 carbonyl, whereas, in large protein system, the protein matrix prohibits such rotation, and thus the plane determined by Arg96 guanidinium is nearly perpendicular to the plane determined by Tyr66 carbonyl. Secondly, in model C, the phenol ring of Tyr66 has rotated and is nearly perpendicular to the fivemembered heterocycle that will be formed (Figure 3), while, in ONIOM-ME case, the phenol ring is nearly in the same plane as the five-membered heterocycle to be formed (Figure 6a), which probably because the protein matrix prohibit the rotation of phenol ring. Accordingly, the rotation of phenol ring in model C leads to that the distance between W2 and carbonyl of Ser65 is shortened, and it is W2 that stabilizes the protonated amide, while it is W1 that stabilizes the protonated amide in ONIOM system.

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In the ONIOM-EE case, the optimized geometries of reactant, transition state and product of this step are listed in Figure 6b. It is shown in Figure 6b that, the distance N67-H67, O65-H67 and C65-O65 are 1.02, 1.94, and 1.25 Å in Re, respectively (Table 3). In TS1, the three distances are 2.40, 0.98 and 1.30 Å respectively. In the proton transfer product Int, the three distances are 3.53, 1.01 and 1.30 Å respectively. The distance between the proton H67 and oxygen of W1 is 1.62 Å. These data show that proton on Gly67 amide nitrogen has been transferred to Ser65 carbonyl oxygen, generating protonated amide, which is stabilized by a hydrogen bond interaction with W1. In particular, the distance between Hη (Arg96) and the O66 is 1.81 Å in Re to 1.80 Å in TS1 and 1.75 Å in Int, which show that Arg96 plays a role of stabilizing the intermediate Int. The energy of TS1 relative to the reactant Re is 33.5 kcal/mol (Figure 7). The intermediate Int is calculated to be 29.6 kcal/mol higher than Re, and this value is 8.6 kcal/mol larger than the corresponding value in ONIOM-ME case, which show that polarization effects by the MM environment is significant on the energy of Int relative to Re. This can also be seen from the differences of the critical geometry parameters between the ONIOM-ME case and the ONIOM-EE case. In detail, in ONIOM-ME case, the distance C65-N67 and Hη-O66 are 3.00 and 1.65 Å respectively in Int, whereas, in ONIOM-EE case, the two distances are 3.09 and 1.75 Å respectively in Int. It can be seen that the distance between Arg96 guanidinium and Tyr66 carbonyl in ONIOM-EE case is large than the corresponding value in ONIOMME case, and accordingly the C65-N67 in ONIOM-EE case is larger than corresponding value in ONIOM-ME case, and accordingly energy barrier of proton transfer in ONIOM-EE case is bigger than the corresponding value in ONIOM-ME case. 3.2.1 Nucleophilic attack Here we will discuss the second step of the cyclization reaction: the Gly67 amide nitrogen attacks the carbonyl carbon of Ser65. In model C, the optimized geometries of nucleophilic attack reactant Int, transition state TS2 and product Pr of this step are listed in Figure 3. The distance C65-N67, C65-O65 and O65-H67 are 2.89, 1.29 and 1.05 Å respectively in Int, and the three distances are 2.17, 1.31 and 1.03 Å respectively in TS2, and 1.51, 1.38 and 1.00 Å respectively in Pr. In addition, the C66-N67 distance is elongated from 1.29 Å in Int to 1.30 Å at TS2 to 1.33 Å at Pr.

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The C66-O66 distance decreases from 1.30 Å in Int to 1.29 Å at TS2 and 1.26 Å at Pr. All these show that the five-membered heterocycle with the hydroxyl attached to it has formed. The Nucleophilic attack transition sate TS2 is identified as the firstorder saddle point with only one imaginary frequency (150.2i cm-1), which corresponds to the C65-N67 stretch mode. The mode is strongly bound to the reaction coordinate, which is supported by IRC analyses. The Nucleophilic attack need overcome only a small barrier, since TS2 lies only 2.0 kcal/mol (4.6 kcal/mol without solvation) higher than Int. The nucleophilic attack product Pr is calculated to be 8.4 kcal/mol (11.0 kcal/mol without solvation) higher than Re (Figure 5), which is in line with the experimental observation that cyclized product is higher in energy than the precyclized chromophore.8 In ONIOM-ME case, the optimized geometries of nucleophilic attack reactant Int, transition state TS2 and product Pr of this step are listed in Figure 6a. The distance C65-N67 is 3.00 Å in Int, and is larger than the corresponding value of model C (2.89 Å). The C65-N67 distance is 2.23 Å at TS2 and 1.49 Å at Pr. The distance C65-O65 and O65-H67 is 1.28 and 1.05 Å in Int, the two distances are 1.31 and 1.04 Å at TS2, and 1.41 and 0.99 Å at Pr. The C66-N67 distance is 1.29 Å in Int to 1.30 Å at TS2 and 1.34 Å at Pr. The C66-O66 distance is 1.29 Å in Int to 1.28 Å at TS2 and 1.25 Å at Pr. All these show that the five-membered heterocycle with a hydroxyl attached to it has formed. The barrier of nucleophilic attack in ONIOM-ME case is 2.1 kcal/mol (Figure 7), which is similar to the value of model C (2.0 kcal/mol). The nucleophilic attack product Pr is calculated to be 5.0 kcal/mol higher than Re. In ONIOM-EE case, the optimized geometries of nucleophilic attack reactant Int, transition state TS2 and product Pr of this step are listed in Figure 6b. The C65-N67 distance is 3.09 Å in Int, and is larger than the result in ONIOM-ME case, which is in step with that the Hη-O66 distance in ONIOM-EE case is larger than the corresponding value in ONIOM-ME case. The C65-N67 distance is 2.23 Å at TS2 and 1.48 Å at Pr. The distance C65-O65 and O65-H67 are 1.30 and 1.01 Å respectively in Int, and the two distances are 1.32 and 1.01 Å at TS2, and are 1.43 and 0.98 Å at Pr. In addition, the C66-N67 distance is 1.30 Å in Int to 1.31 Å at TS2 and 1.35 Å at Pr. The C66-O66 distance is 1.27 Å in Int to 1.27 Å at TS2 and 1.24 Å at Pr. All these show that the five-membered heterocycle with a hydroxyl attached to

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it has formed. The energy barrier of nucleophilic attack in ONIOM-EE case is also small, and is 5.2 kcal/mol (Figure 7). The nucleophilic attack product Pr is calculated to be 10.5 kcal/mol endothermic, which is in line with experimental observation.8 4. Conclusion Cyclization in chromophore maturation of wild-type GFP is not a one-step process that the amide nitrogen of Gly67 attacks Ser65 carbonyl carbon directly, and simultaneously the proton on Gly67 amide nitrogen transfers to Ser65 carbonyl oxygen, resulting in the formation of five-membered heterocycle with a hydroxyl attached to it. A possible two-step mechanism of cyclization is investigated: first, proton on amide nitrogen of Gly67 transfers to Ser65 carbonyl oxygen forming protonated amide; second, amide nitrogen of Gly67 attacks the carbonyl carbon of Ser65. The crystal water molecules play an important part in stabilizing the intermediate protonated amide, so that the intermediate could exist stably. The intermediate we found is a candidate for a biosynthetic study as a possible imitation of GFP. The Arg96 has contribution to stabilization of the intermediate. The protein environment, especially the hydrogen bond interactions around the precyclized chromophore play important roles in the two-step cyclized mechanism. The cyclization reaction is calculated to be endothermic by about 10 kcal/mol, which is in agreement with experimental results that cyclized product has the low thermostability. The two-step mechanism has the lowest energy barrier in all the proposed mechanisms of cyclization in chromophore maturation of wild-type GFP so far.

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 (No. ZD201420), and supported by Program for Innovative Research Team in Universities of Inner Mongolia Autonomous Region (No. NMGIRT-A1603), and network center of Inner Mongolia University of Technology.

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Reference: (1) Remington, S. J. Fluorescent Proteins: Maturation, Photochemistry and Photophysics. Curr. Opin. Struct. Biol. 2006, 16, 714-721. (2) Lemay, N. P.; Morgan, A. L.; Archer, E. J.; Dickson, L. A.; Megley, C. M.; Zimmer, M. The Role of the Tight-Turn, Broken Hydrogen Bonding, Glu222 and Arg96 in the Post-Translational Green Fluorescent Protein Chromophore Formation. Chem. Phys. 2008, 348, 152-160. (3) Heim, R.; Prasher, D. C.; Tsien, R. Y. Wavelength Mutations and Posttranslational Autoxidation of Green Fluorescent Protein. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 1250112504. (4) Crameri, A.; Whitehorn, E. A.; Tate, E.; Stemmer, W. P. C. Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling. Nat. Biotechnol. 1996, 14, 315-319. (5) Cubitt, A. B.; Heim, R.; Adams, S. R.; Boyd, A. E.; Gross, L. A.; Tsien, R. Y. Understanding, Improving and Using Green Fluorescent Proteins. Trends Biochem. Sci. 1995, 20, 448-455. (6) Per, E. M. S.; Maria, W.; Marc, Z. Theoretical Study of the Mechanism of Peptide Ring Formation in Green Fluorescent Protein. Int. J. Quantum Chem. 2001, 81, 169-186. (7) Barondeau, D. P.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. Understanding GFP Posttranslational Chemistry: Structures of Designed Variants that Achieve Backbone Fragmentation, Hydrolysis, and Decarboxylation. J. Am. Chem. Soc. 2006, 128, 4685-4693. (8) Barondeau, D. P.; Putnam, C. D.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. Mechanism and Energetics of Green Fluorescent Protein Chromophore Synthesis Revealed by Trapped Intermediate Structures. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 12111-12116. (9) Barondeau, D. P.; Tainer, J. A.; Getzoff, E. D. Structural Evidence for an Enolate Intermediate in GFP Fluorophore Biosynthesis. J. Am. Chem. Soc. 2006, 128, 3166-3168. (10) Wood, T. I.; Barondeau, D. P.; Hitomi, C.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. Defining the Role of Arginine 96 in Green Fluorescent Protein Fluorophore Biosynthesis. Biochemistry 2005, 44, 16211-16220. (11) Barondeau, D. P.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. The Case of the Missing Ring: Radical Cleavage of a Carbon-Carbon Bond and Implications for GFP Chromophore Biosynthesis. J. Am. Chem. Soc. 2007, 129, 3118-3126. (12) Barondeau, D. P.; Kassmann, C. J.; Tainer, J. A.; Getzoff, E. D. Understanding GFP Chromophore Biosynthesis: Controlling Backbone Cyclization and Modifying Posttranslational Chemistry. Biochemistry 2005, 44, 1960-1970. (13) Sniegowski, J. A.; Lappe, J. W.; Patel, H. N.; Huffman, H. A.; Wachter, R. M. Base Catalysis of Chromophore Formation in Arg96 and Glu222 Variants of Green Fluorescent Protein. J. Biol. Chem. 2005, 280, 26248-26255. (14) Sniegowski, J. A.; Phail, M. E.; Wachter, R. M. Maturation Efficiency, Trypsin Sensitivity, and Optical Properties of Arg96, Glu222, and Gly67 Variants of Green Fluorescent Protein. Biochem. Biophys. Res. Commun. 2005, 332, 657-663. (15) Wachter, R. M. Chromogenic Cross-Link Formation in Green Fluorescent Protein. Acc. Chem. Res. 2007, 40, 120-127. (16) Wachter, R. M. Mechanistic Aspects of GFP Chromophore Biogenesis, Genetically Engineered Probes for Biomedical Applications, San Jose, CA, USA, Jan 22-24, 2006; Savitsky A. P., Wachter, R. M., Eds.; SPIE: Bellingham, 2006. (17) Zhang, L.; Patel, H. N.; Lappe, J. W.; Wachter, R. M. Reaction Progress of Chromophore Biogenesis in Green Fluorescent Protein. J. Am. Chem. Soc. 2006, 128, 4766-4772. (18) Rosenow, M. A.; Huffman, H. A.; Phail, M. E.; Wachter, R. M. The Crystal Structure of the Y66L Variant of Green Fluorescent Protein Supports a Cyclization-Oxidation-Dehydration Mechanism for Chromophore Maturation. Biochemistry 2004, 43, 4464-4472. (19) Pouwels, L. J.; Zhang, L.; Chan, N. H.; Dorrestein, P. C.; Wachter, R. M. Kinetic Isotope

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Effect Studies on the De Novo Rate of Chromophore Formation in Fast- and Slow-Maturing GFP Variants. Biochemistry 2008, 47, 10111-10122. (20) Rosenow, M. A.; Patel, H. N.; Wachter, R. M. Oxidative Chemistry in the GFP Active Site Leads to Covalent Cross-Linking of a Modified Leucine Side Chain with a Histidine Imidazole: Implications for the Mechanism of Chromophore Formation. Biochemistry 2005, 44, 83038311. (21) Ma, Y.; Yu, J.-G.; Sun, Q.; Li, Z.; Smith, S. C. The Mechanism of Dehydration in Chromophore Maturation of Wild-Type Green Fluorescent Protein: A Theoretical Study. Chem. Phys. Lett. 2015, 631–632, 42-46. (22) Ma, Y.; Sun, Q.; Zhang, H.; Peng, L.; Yu, J.-G.; Smith, S. C. The Mechanism of Cyclization in Chromophore Maturation of Green Fluorescent Protein: A Theoretical Study. J. Phys. Chem. B 2010, 114, 9698-9705. (23) Ma, Y.; Sun, Q.; Li, Z.; Yu, J.-G.; Smith, S. C. Theoretical Studies of Chromophore Maturation in the Wild-Type Green Fluorescent Protein: ONIOM(DFT:MM) Investigation of the Mechanism of Cyclization. J. Phys. Chem. B 2012, 116, 1426-1436. (24) Trujillo, C.; Sánchez-Sanz, G.; Alkorta, I.; Elguero, J. A Theoretical Investigation of the Mechanism of Formation of a Simplified Analog of the Green Fluorescent Protein (GFP) from a Peptide Model. Struct. Chem. 2013, 24, 1145-1151. (25) Becke, A. D. Density-functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648-5652. (26) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-energy Formula into a Functional of the Electron Density. Phys. Rev. B 1988, 37, 785-789. (27) Schäfer, A.; Huber, C.; Ahlrichs, R. Fully Optimized Contracted Gaussian Basis Sets of Triple Zeta Valence Quality for Atoms Li to Kr. J. Chem. Phys. 1994, 100, 5829-5835. (28) Gonzalez, C.; Schlegel, H. B. An Improved Algorithm for Reaction Path Following. J. Chem. Phys. 1989, 90, 2154-2161. (29) Gonzalez, C.; Schlegel, H. B. Reaction Path Following in Mass-weighted Internal Coordinates. J. Phys. Chem. 1990, 94, 5523-5527. (30) Barone, V.; Cossi, M. Quantum Calculation of Molecular Energies and Energy Gradients in Solution by a Conductor Solvent Model. J. Phys. Chem. A 1998, 102, 1995-2001. (31) Cammi, R.; Mennucci, B.; Tomasi, J. Second-Order Møller-Plesset Analytical Derivatives for the Polarizable Continuum Model Using the Relaxed Density Approach. J. Phys. Chem. A 1999, 103, 9100-9108. (32) Vreven, T.; Byun, K. S.; Komáromi, I.; Dapprich, S.; Montgomery, J. A.; Morokuma, K.; Frisch, M. J. Combining Quantum Mechanics Methods with Molecular Mechanics Methods in ONIOM. J. Chem. Theory Comput. 2006, 2, 815-826. (33) Humbel, S.; Sieber, S.; Morokuma, K. The IMOMO Method: Integration of Different Levels of Molecular Orbital Approximations for Geometry Optimization of Large Systems: Test for n-butane Conformation and SN2 Reaction: RCl+Cl-. J. Chem. Phys. 1996, 105, 1959-1967. (34) Svensson, M.; Humbel, S.; Froese, R. D. J.; Matsubara, T.; Sieber, S.; Morokuma, K. ONIOM: A Multilayered Integrated MO + MM Method for Geometry Optimizations and Single Point Energy Predictions. A Test for Diels−Alder ReacJons and Pt(P(t-Bu)3)2 + H2 Oxidative Addition. J. Phys. Chem. 1996, 100, 19357-19363. (35) Maseras, F.; Morokuma, K. IMOMM: A New Integrated Ab Initio + Molecular Mechanics Geometry Optimization Scheme of Equilibrium Structures and Transition States. J. Comput. Chem. 1995, 16, 1170-1179. (36) Dapprich, S.; Komáromi, I.; Byun, K. S.; Morokuma, K.; Frisch, M. J. A New ONIOM Implementation in Gaussian98. Part I. The Calculation of Energies, Gradients, Vibrational Frequencies and Electric Field Derivatives. THEOCHEM 1999, 461-462, 1-21. (37) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.;

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Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A., et al. Gaussian 09, Revision C.01; Gaussian, Inc., Wallingford, CT, 2010.

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Page 16 of 28

Table 1. Important geometry parameters (in Å) in the two-step reaction path calculated with model C.

Model C Distance(Å)

Re

TS1

Int

TS2

Pr

C(Ser65)…N(Gly67)

3.12 3.28 2.89 2.17 1.51

N(Gly67)…H(Gly67)

1.03 2.25 3.72 3.25 2.77

H(Gly67)…O(Ser65)

1.71 1.00 1.05 1.03 1.00

C(Ser65)…O(Ser65)

1.25 1.30 1.29 1.31 1.38

C(Tyr66)…O(Tyr66)

1.26 1.30 1.30 1.29 1.26

C(Tyr66)…N(Gly67)

1.33 1.29 1.29 1.30 1.33

Hη(Arg96)…O(Tyr66)

1.75 1.60 1.54 1.67 1.75

Oε1(Glu222)...Hγ(Ser65)

1.71 1.69 1.75 1.75 1.75


2.68 2.75 2.82 2.77 2.57

Hα(Tyr66)...O(H2O1058)

2.08 2.22 2.38 2.55 3.21

H(H2O1058)...O(H2O1042) 1.90 1.99 1.80 1.82 1.75 H(H2O1042)... Oε2(Glu222) 1.87 1.78 1.73 1.71 1.63 H(Gly67)…O(H2O1058)

3.84 1.94 1.48 1.53 1.68

Oη(Tyr66)…Hε(His148)

1.92 1.92 1.94 1.92 1.89

Hη(Tyr66)...Oγ(Ser205)

1.67 1.68 1.71 1.70 1.67


1.62 1.66 1.66 1.64 1.60

16


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Table 2. Important geometry parameters (in Å) in the two-step reaction path calculated in ONIOM-ME case.

ONIOM-ME Distance(Å)

Re

TS1

Int

TS2

Pr

C(Ser65)…N(Gly67)

3.08 3.02 3.00 2.23 1.49

N(Gly67)…H(Gly67)

1.02 2.30 3.75 3.42 2.99

H(Gly67)…O(Ser65)

1.95 0.98 1.05 1.04 0.99

C(Ser65)…O(Ser65)

1.24 1.30 1.28 1.31 1.41

C(Tyr66)…O(Tyr66)

1.25 1.29 1.29 1.28 1.25

C(Tyr66)…N(Gly67)

1.34 1.29 1.29 1.30 1.34


1.67 1.64 1.65 1.68 1.75


1.75 1.73 1.90 2.81 2.67


2.50 2.50 2.49 1.74 1.62

Hα(Tyr66)...O(H2O1058)

2.23 2.53 2.51 2.34 2.29

H(H2O1058)...O(H2O1042) 1.73 1.83 1.93 1.94 1.88 H(H2O1042)... Oε2(Glu222) 1.74 1.69 1.43 1.43 1.61 H(Gly67)…O(H2O1042)

4.12 2.42 1.47 1.49 1.77


1.93 1.93 1.93 1.90 1.89


2.00 2.03 2.03 1.92 1.87


1.62 1.64 1.61 1.53 1.52

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Page 18 of 28

Table 3. Important geometry parameters (in Å) in the two-step reaction path calculated in ONIOM-EE case.

ONIOM-EE Distance(Å)

Re

TS1

Int

TS2

Pr

C(Ser65)…N(Gly67)

3.10

3.09 3.09 2.23 1.48

N(Gly67)…H(Gly67)

1.02

2.40 3.53 3.31 2.96

H(Gly67)…O(Ser65)

1.94

0.98 1.01 1.01 0.98

C(Ser65)…O(Ser65)

1.25

1.30 1.30 1.32 1.43

C(Tyr66)…O(Tyr66)

1.23

1.27 1.27 1.27 1.24

C(Tyr66)…N(Gly67)

1.35

1.31 1.30 1.31 1.35


1.81 1.80 1.75 1.77 1.92


1.79

1.78 1.88 2.76 2.56


2.49

2.50 2.50 1.77 1.70

Hα(Tyr66)...O(H2O1058)

2.37

2.66 2.65 2.44 2.41

H(H2O1058)...O(H2O1042)

1.73

1.82 1.90 1.94 1.87

H(H2O1042)... Oε2(Glu222)

1.75

1.65 1.49 1.49 1.64

H(Gly67)…O(H2O1042)

3.99

2.31 1.62 1.62 1.89


1.91

1.93 1.94 1.91 1.89


1.88

1.87 1.87 1.81 1.77


1.69

1.71 1.70 1.63 1.61

18


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Figure captions Figure 1. Two different kinds of reaction scheme of cyclization in chromophore maturation of wild-type GFP: one-step mechanism (a), two-step mechanism (b). Figure 2. Optimized geometries of the reactant, transition state and product of model A and model B system, respectively. Figure 3. Optimized geometries of the reactant, transition states, intermediate and product of model C system. Figure 4. The potential energy profile of one-step mechanism of model A and model B, respectively. a: the energies of b3lyp/6-311+G(2d,2p) level with zero-point correction, b: the energies of CPCM (ε=4). Figure 5. The potential energy profile of two-step mechanism of model C system. Figure 6. Optimized geometries of the reactant, transition states, intermediate and product by ONIOM-ME (a) and ONIOM-EE (b) respectively. For the sake of clarity, some atoms are omitted. Figure 7. Potential energy profile of two-step mechanism of large protein system, energies

of

ONIOM(b3lyp/6-31G(d,p):Amber)

corresponding

to

geometries

optimized by mechanical embedding and electronic embedding respectively.

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Page 20 of 28

O

O

Tyr66

Gly67 N HN

N

H O

O

HN

HO

Ser65

OH

HO

HO NH

(a) O

Tyr66

HO

HN HO Ser65

O

Gly67 N

N

H

O

HO

NH

O

HN HO

OH

N HO

NH

(b)

Figure 1

20


HN

OH

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2.25 0.99 1.34

3.68

A-Re

A-TS

1.47 0.97 1.41

A-Pr

(a)

Arg96 1.86

Arg96

Arg96

1.86

1.74

3.19

2.37

1.50 0.97

1.4 0.97 0

1.32

B-Re

B-TS

(b)

Figure 2

21


B-Pr


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

*

* Arg96

Arg96

1.75

*

1.33

1.25

1.71

1.67

1.92

2.03

1.66

Ser205

Glu222

Glu222 *

*

Re

1.30

1.69

1.68

*

TS1 *

*

Arg96

Arg96

1.67

1.54

*

*

1.29

1.30

1.30

1.29

2.89 1.29

2.25 1.00

His148

1.87

1.62

Ser205 *

1.29

1.03 1.71

1.92

His148

1.60 1.30

*

1.26

2.17

3.72 His148

1.94

1.0 5 1.48

1.31

1.71 Ser205

1.75 Glu222

1.03

1.53

1.92 1.70

1.75 Ser205

*

* *

Int

TS2 *

Arg96

1.75 1.26 *

1.33

1.51 1.38 1.00 1.68

1.7

1.89 5

1.60

Glu222 *

His148

1.67 Ser205 *

Pr Figure 3

22


Glu222 *

His148

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Relative Energies (kcal/mol)

Page 23 of 28

A-TS

60 50

B-TS

40

40.2a 43.2b

30

B-Pr

17.0a 16.1b

20 10 0

Model A Model B

49.6ab 50.8

A-Pr

A-Re

13.6a 13.9b

0.0 0.0

B-Re

Figure 4

23



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30.9 27.6

29.6 24.0

23.4 19.4

11.0 8.4 0.0 0.0 Re

TS1

Int

TS2

Pr

Figure 5

24


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Arg96

Arg96 1.77

1.67

3.02 2.30

1.02 3.08 1.95

0.98

1.75

1.73

Glu222

Glu222

Re

TS1

Arg96 1.65

1.75

1.68

3.00

1.49

2.23 1.05

1.47

1.90

Glu222

Int

Arg96

Arg96

1.04 1.49 1.74

Glu222

TS2

(a)

25


0.99 1.77 1.62

Glu222

Pr


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

Arg96

Arg96

1.82

1.81

3.09

3.10

2.40

1.94

0.98 1.78

1.79

Glu222

Glu222

Re

TS1

Arg96

Arg96

Arg96

1.92

1.77

1.75

1.48

2.23

3.09

0.98

1.01

1.01 1.62 1.88

Glu222

1.62

1.70

1.77

Glu222

Int

1.89

TS2

(b)

Figure 6

26


Glu222

Pr

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33.5 29.4

34.8 29.6 21.0

23.1

10.5 5.0

0.0 0.0 Re

TS1

Int

TS2

Pr

Figure 7

27



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Table of Contents

28


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New Insights on the Mechanism of Cyclization in Chromophore

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