Peptide Bond trans–cis Isomerization and Acylimine Formation in

Aug 11, 2011 - Yarbrough , D.; Wachter , R. M.; Kallio , K.; Matz , M. V.; Remington , S. J. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 462– 467. [Cros...
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Peptide Bond trans cis Isomerization and Acylimine Formation in Chromophore Maturation of the Red Fluorescent Proteins Xuefeng Ren,† Daiqian Xie,*,† and Jun Zeng*,‡,§ †

Institute of Theoretical and Computational Chemistry, Key Laboratory of Mesoscopic Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, People’s Republic of China ‡ College of Chemistry, Sichuan University, Chengdu 610064, People’s Republic of China § MedChemSoft Solutions, P.O. Box 5143, Wantirna South, 3152, Australia ABSTRACT: In red fluorescent proteins such as DsRed, an acylimine is formed from the Phe65-Gln66 linkage in GFP-like immature form, while it shows a cis configuration in its mature state. To date, the relationship between acylimine formation and trans cis isomerization is still unresolved. We have calculated bond rotation profiles for mature and immature chromophores within the protein using our own n-layered integrated molecular orbital and molecular mechanism (ONIOM) approach. The results suggested that the isomerization is barrierless in acylimine formed in the mature state, suggesting that the acylimine formation precedes the trans cis isomerization in DsRed chromophores. Further decomposition analysis of electrostatic contributions from individual residues has identified several residues and a specific water molecule which could play key roles in controlling the rate of the trans cis isomerization of peptide bond in immature GFP-like protein. The results also highlight the importance of Gln66-like of tripeptide motif (chromophore) in the maturation of red fluorescent proteins. In view of the considerable interest in developing red fluorescent proteins for numerous biotechnological applications, these results should be useful for design of novel fluorescent proteins.

1. INTRODUCTION Since the discovery of GFP,1 fluorescent proteins have been widely used in life-science researches as molecular labels, noninvasive markers of gene expression, and reporters of environmental conditions within living cells.2 7 Three-dimensional structures of the 238-amino acid GFPs show a perfect β-barrel fold symmetry and at the center of the protein lies a chromophore that is responsible for the observed fluorescence.8 The unusual p-hydroxylbenzylideneimidazolinone chromophore is formed by autocatalytic post-translational cyclization of tripeptide Ser65-Tyr66-Gly67, which is well protected from the solvent by the β-barrel.9 In wild-type GFP, two broad absorption bands, at 397 and 477 nm, have been assigned to the lowest singlet excitation of the chromophore at neutral and anionic protonation states, respectively.10,11 In recent years, some novel fluorescent proteins with red fluorescence emission have also been identified, complementing the existing GFP technology.12 The first of these, DsRed, is a 28 kDa protein with bright red fluorescence, which is isolated from a corallimorpharian of the Discosoma genus.13 Crystallographic studies showed that DsRed has a chromophore (tripeptide Gln66-Tyr67-Gly68), which is essentially the same as that found in the GFPs, except for an extension of the conjugate π system by formation of an acylimine between the CR and N atoms of Gln66.12,14,15 Theoretical calculations demonstrated that formation of this acylimine is responsible for the large red shift observed in the absorption spectrum of DsRed.14 r 2011 American Chemical Society

The proposed mechanism of the autocyclization of the peptide in GFP and its derivatives such as DsRed involves three steps: the peptide cyclization is first initiated by nucleophilic attack of the Gly67 amide-nitrogen on the Ser65 (Gln66 in DsRed) carbonyl-carbon to form a five-member imidazoline ring, followed by a dehydration of the Ser65 carbonyl oxygen and then a rate-limiting oxidation of the Tyr66 (Tyr67 in DsRed) CR Cβ bond to conjugate the ring systems.2,14,16,17 For the mature red fluorescent protein, such as DsRed, the chromophore undergoes a further oxidation reaction that results in a double bond between the Gln66 N and CR atoms.14 This is evident from the spectroscopic observation that the wild-type DsRed also contains greenemitting species;12,18 22 during the course of the protein maturation, the green emission decreases at the same rate as the red fluorescence grows.18,19 Recent crystallographic study has confirmed that the green emission is due to the electronic excitations of the GFP-like chromophore in DsRed and its mutants.23 While the immature form of DsRed has been considered to contain a trans peptide bond, the trans cis isomerization was traditionally considered not to be responsible for the green to red maturation of DsRed.12,24 However, recent crystallographic studies on the immature and mature forms of DsRed and their mutants demonstrated that the immature and mature DsRed chromophores adopt a trans and cis peptide bond, respectively, and the acylimine formation is linked to the peptide bond Received: April 11, 2011 Revised: August 5, 2011 Published: August 11, 2011 10129

dx.doi.org/10.1021/jp2033609 | J. Phys. Chem. A 2011, 115, 10129–10135

The Journal of Physical Chemistry A

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Table 1. Important Bond Lengths, Angles, and Torsional Angles around the Acylimine Groups of the Chromophore Optimized at Immature (G) and Mature (R) States of Red Fluorescent Protein DsRed, Based on Its Crystal Structures23 (PDB code: 1ZGO) immature (G)

Figure 1. Optimized structures of the chromophore from the crystal structure (1ZGO) of DsRed protein.23 Upper panel shows the chromophore structure with the important atoms labeled. Lower panel shows the chromophore within DsRed protein. The results were obtained at ONIOM(B3LYP/6-31+G(d,p):Amber) level.

isomerization.12,24,25 The mechanism of coupling between the peptide bond isomerization and acylimine formation is still not well established,26 especially as to how the surrounding protein environment affects the processes of the isomerization and acylimine formation. In this work, we have explored the potential energy landscape along a dihedral reaction coordinate for the trans cis isomerization reaction of the immature and mature chromophore in the fluorescent protein DsRed. The energetic decomposition of contribution from each amino acid to the energy barrier of the isomerization reaction was also analyzed. This will bring some useful insights into our understanding of the chromophore formation and maturation and suggest mutants for engineering new red fluorescent proteins.27

2. METHODS The initial structures of the DsRed was taken from the Protein Data Bank (PDB ID: 1ZGO23) for both immature (G) and mature (R) forms of chromophore, which are protonated as an anionic state, respectively. Hydrogen atoms were added with the Gaussview program and optimized at the Molecular Mechanical (MM) level with Amber force field parameters.27 For coordinatedriving scans of the potential energy surface, we optimized the chromophore under the constraint of a constant dihedral angle ϕ(C1 N1 CR O1) (see Figure 1 for the dihedral definition and atom numbering). In general, medium effects from the surrounding protein environment on the energetic and spectroscopic properties of the chromophore can be treated using either the dielectric continuum model (e.g., a self-consistent reaction-field),43 our own n-layered integrated molecular orbital and molecular mechanics (ONIOM) approach,28 or the combined quantum mechanical (QM)/molecular mechanical (MM) simulation methods.44 While the dielectric continuum model is too simplified for the GFP and RFP chromophores, the QM/MM molecular dynamics simulations are too computationally expensive.45 We thus used the ONIOM approach with the B3LYP method32 34 and 6-311+G(d,p) basis set35,36 and to mimic the electrostatic

mature (R)

CR N1a

1.38

1.35

CR Ra CR O1a

1.32 1.23

1.33 1.25

N1 C1a

1.48

1.27

R CR N1b

122.7

118.8

N2 C2 C1b

123.6

124.0

C2 C1 N1b

109.3

115.5

C1 N1 CRb

110.6

ϕ(C1 N1 CR O1)c

14.4d

Φ(N2 C2 C1 N1)c

147.8

154.0 129.9d 169.7

a

Bond length in Å. b Bond angle in degrees. c Torsion angle in degrees. d Experimental values of 5.6 and 164.6°.23

effects of the surrounding protein environment on the chromophore. The quantum mechanical (QM) part consists of the chromophore formed by the tripeptide (Gln66-Tyr67-Gly68), two surrounding residues (Phe65 and Ser69), a water molecule, and two linking H atoms (Figure 1). The chromophore is protonated at anionic state for both the green immature and red mature forms of the DsRed, respectively. The rest of the protein was treated as the molecular mechanical (MM) part using an Amber force field.27 All the optimizations were performed at the ONIOM (B3LYP/6-311+G(d,p): Amber) level with electronic embedding.37 We also performed decomposition analysis29 31 of the energy barrier for the trans cis isomerization. To analyze the electrostatic contributions of individual residues, the energies of the optimized trans isomers and transition states of the immature and mature chromophores were recomputed with atomic charges from all residues switched off, except for the residue of interest.29 31 This procedure was applied to all residues and water molecules bound to the systems. All the calculations were performed using Gaussian 03 program.38

3. RESULTS The initial structures of immature and mature chromophores within the red fluorescent protein were first optimized using ONIOM method at B3LYP/6-31+G(d,p) level. Figure 1 shows the optimized structures of the chromophores, and Table 1 gives values for the most important variables for the peptide bond and acylimine group of the chromophore at immature state G and mature state R. Overall, the geometrical structures of the chromophores during the maturation differ mainly at the acylimine part. While the chromophore at the immature state (G) has the peptide bond in a tetrahedral geometry with bond angle R CR N1 of 122.7°, the acylimine form of peptide bond at the mature state (R) adopts a triangular conformation with the bond angle of 118.8°. The bond distance of CR N1, CR R, and CR O1 are calculated to be 1.38, 1.32, and 1.23 Å at the state G and 1.35, 1.33, and 1.25 Å at the state R, respectively. Around the peptide bond/acylimine group, the bond length of N1 C1 is 1.48 and 1.27 Å for the G and R states, respectively, indicating that the N1 C1 forms a double bond in the mature protein. 10130

dx.doi.org/10.1021/jp2033609 |J. Phys. Chem. A 2011, 115, 10129–10135

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Figure 2. Energy profiles of the chromophore along the reaction coordinates of torsional angle ϕ(C1 N1 CR O1) for immature state G (A) and mature state R (B), together with the chromophore structures at the trans conformation, transition state, and cis conformation. The values of the important variables are given in Table 2. All the results were obtained at ONIOM(B3LYP/6-31+G(d,p):Amber) level.

The torsion angle of ϕ(C1 N1 CR O1) is predicted to be 14.4 and 129.9° for the immature and mature chromophores, indicating trans and cis conformations for the corresponding peptide bond and acylimine groups, respectively. While the torsional angle for the peptide bond of immature chromophore is consistent with the experimental value of 5.6° (1ZGO),23 the optimized torsional angle for the acylimine group is 35° less than the value observed in the X-ray structures (1ZGO).23 This could be due to the specific water molecule in the QM part which was found to form hydrogen bond with the carbonyl group of acylimine. A smaller torsional angle (153.3°) was also observed from the recent crystal structure of eqFP611 in which a water molecule was found to be 2.91 Å away from the acylimine-carbonyl.46 Note that during the optimization, the residues Phe65 and Ser69 that are linked to the chromophore and treated as QM part in the ONIOM calculations do not display any significant movement from their initial positions at the crystal structures. Figure 2 shows the energy profile of the trans cis isomerization of the peptide bond in the immature state G and of the acylimine at the mature state R of the chromophore. Torsion angle ϕ(C1 N1 CR O1) was used as the reaction coordinate. Starting from the trans conformation of the peptide bond/ acylimine of the chromophores, the torsion angle was rotated at intervals of 30° and kept fixed during the geometry optimization. When the torsion angle reached 180°, the peptide bond

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(or acylimine in R) was found to adopt the cis conformation. Beyond 180° at the immature state and 240° (or 120°) at the mature state, the CR O1 group rotates toward the chromophore part linked to Ser69, resulting in a large increase in total energy (Figure 2). The minimum of cis conformation is thus predicted to be at the torsional angle C1 N1 CR O1 of 180° for the state G and 230.1° (or 129.9°) for the state R. The latter is consistent with the results obtained from geometry optimization from the crystal structure as described above. From the profile, the transition states of the isomerization were identified at the torsional angle ϕ(C1 N1 CR O1) of about 75 and 90° for the immature and mature chromophores, respectively. Figure 2 also shows all the structures of the trans isomer (Trans), transition states (TS) and cis isomer (Cis) of the chromophores, and Table 2 lists all the important variables, energy barrier, as well as relative energies of the isomerization. Upon the trans cis isomerization of the peptide bond at the state G, noticeable conformational changes occur at the peptide bond and its linked residue Phe65 as the change in torsion angle shifts the backbone of Phe65 (e.g., amide N) by up to 1.35 Å at the transition state (TS/G). For the mature state R, however, the conformational changes become negligible as the position of the atom N1 or C1 of acylimine group shifts by less than 0.3 Å during the isomerization. As a result, the trans and cis isomers of the chromophore were predicted to be equivalently stable (relative energies of 0.84 kcal/mol) and the isomerization is barrierless with the energy barrier estimated to be