QM/MM Study of the Second Harmonic Generation and Two-photon Absorption Properties of aFluorescent Proteins-Dreiklang①

ZHANG Min-Yi WEI Jing SONG Jin-Shui WU Peng LI Chun-Sen

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QM/MM Study of the Second Harmonic Generation and Two-photon Absorption Properties of aFluorescent Proteins-Dreiklang①

ZHANG Min-Yia, bWEI JingaSONG Jin-Shuaia, bWU PengaLI Chun-Sena, b②

a(350002)b(361005)

A new reversibly switchable fluorescent protein (RSFP), namely Dreiklang, exhibits prominent feature that the wavelengths for switching and fluorescence are decoupled due to its great different structures between bright and dark states.This feature might also induce some nonlinear optic (NLO) properties changing as switching between two states, which might promote new method of biological science.We employ the QM/MM method to simulate the structures of different states, and study their second harmonic generation (SHG) and two-photon absorption (TPA) properties.And we found different states of Dreiklang have different SHGand TPA responses.TheSHGand TPA properties of Dreiklang are correlated to particularly geometrical structures of different states, especially the centrosymmetric or nocentrosymmetric-stacking structures which are formed by chromophore and beside residue Tyr203, so the SHGand TPA responses could be changed as the light induces switching among different states of Dreiklang.This work would prospectively guide the application of Dreiklang on the NLO technology, and help the development of new RSFP with special NLO function.

fluorescent proteins,the second harmonic generation, two-photon absorption, QM/MM method;

1 INTRODUCTION

Fluorescent Proteins (FPs) have been widely used in cell biology like molecular labels, noninvasive markers of gene expression, and intracellular protein localization etc[1,2].Recently, a new kind of FPs namely reversibly switchable fluorescent proteins (RSFPs) has attracted great attention[3,4].These proteins could be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, which results in the controllable emitting fluorescent on-to-off transition[5].Due to this character, RSFPs have been used for super-resolution microscopy, fluorescence correlation spectroscopy, optical lock-in detection, and biotechnological developments such as rewritable high-density optical storage media[6-9].On the other hand, this particular characters of RSFPs evoke an exciting speculation that some RSFPs might exhibit the on-to-off transition of NLO properties, since repeatedly off/on switching of RSFPs is mainly attributed to the structural rearrange-ment in different states and the NLO properties are depending on the materials’ structure[10,11].

It is well known that nonlinear optical (NLO) pro- perties, such as the second harmonic generation(SHG)[12]and two-photon absorption (TPA)[13], are under intense investigation due to many important applications like optical bistability, phase conjuga- tion, optical limiting, two-photon laser scanning microscopy and so on[14-16].Owing to the advantages used in live cell, FPs haveattracted much attention on the NLO biological imaging in the past decade[17,18].And Asselberghs and his coworkers have studied the SHG properties of a RSFPs named Dronpa, findingits SHG propertiescould be changed accor- ding to the protonation of chromophore structure[19].This work illuminated the potentially NLO photoswitchable properties of RSFPs.

Recently, Brakemann and his coworkers have developed new RSFPs named Dreiklang from a kind of YFP namely Citrine[20].Dreiklang exhibits two absorption bands (peaking at 412 and 511 nm) in its on-state, corresponding to the neutral (protonated) and ionized (deprotonated) states of the chromophore, respectively, both of which emit 529nm fluorescent light by excitation; its off state presents a 340nm absorption band, which corresponds to the structural rearrangement of chromophore through light-induce hydration of the imidazolinone ring from the on-state.The structural rearrangement between the on- and dark-states of Dreiklang would changethe transition moments and energies, and these features mainly dominate the NLO properties of Dreiklang.Interestly, Beerepoot and coworkers studied the TPA properties of YFP, and they found that the-stacking structure of chromophore and the tyrosine 203 could enhance the TPA response of YFP[21].Furthermore, Clays and coworkers developed a new Tyr203Phe mutant of eYFP, whose SHG properties were enhanced through Tyr203Phe mutant to remove the centrosymmetric stacking of the chromophoric Tyr66 and the neighboring Tyr203 residue in eYFP[22].

As Dreiklang was a mutant from a kind of YFP namely Citrine, its protonated chromophoric Tyr66 and Tyr203 residues of on state also form the centrosymmetric-stacking structure as YFP, which was determinated by X-ray crystallography(PDB code: 3NT9)[20].Therefore,the light induce swi- tchable feature among on-off states of Dreiklang absolutely influents this centrosymmetric-stacking structure, which might result in the change of SHG and TPA responses in Dreiklang.To further under- stand these particular NLO switchable properties of Dreiklang, in this work, we employ the QM/MM method to simulate different states of Dreiklang, and study their SHG and TPA properties.To the best of our knowledge, it is the first time to propose the protonated form of Dark state chromophore in Dreiklang, and reveal the structure-depending NLO switchable properties on Dreiklang.This work might help to guide the development of new RSFP with NLO switchable function, and develop new application of RSFP on biological imaging techno- logy.

2 COMPUTATIONAL METHODS

2.1 MD runs

The initial structures of Brightand Dark states of Dreiklang were obtained from the Protein Data Bank (PDB IDs: 3NT9 and 3NT3), respectively[20].And the absent specific force-field parameters of chromophores in these four states were obtained by the cgenff program[23, 24].We assigned the proto- nation states of acidic and basic residues by using the pKa values given by the empirical PROPKA[25]procedure and verified the results through careful visual inspection.The missing hydrogen atoms were added by the HBUILD module[26]embed in CHARMM[27].As both chromophores of the Bright and Dark states might have protonated and deprotonated form, the protonation procedure of 3NT9 and 3NT3 results in four states of Bright neutral state (Bn), Bright anionic state (Ba), Dark neutral state (Dn) and Dark anionic state (Da), as shown in Fig.1.All four states of Dreiklang contain 26971 atoms, including 22104 atoms of solvent.After the full solvation procedure, a productive MD of 2ns was run for three states of Dreiklang using the CHARMM22 force fields implemented in the CHARMM program.The coordinates of the outer 8 ? of solvent layer were kept fixed during all MD simulations.Four representative snapshots from the MD trajectory of Bn, Ba and Dn were selected at 1, 1.5, 1.2 and 1 ns, respectively.

2.2 QM/MM methodology

The QM region in our QM/MM calculations including chromophore, residue Glu222, residue Tyr203 and Wat242 is shown in Fig.1.All the geometry optimization calculations were carried out within ChemShell[28], combining Gaussian03[29]as QM code and DL_POLY[30]as MM code.The QM/MM boundary was treated by the hydrogen link atom[31]with the charge shift model[32].For the QM region, the B3LYP[33-35]functional was employed with 6-31G* for geometry optimization.The HDLC optimizer[36]was employed in the geometry optimization and the core regions of optimization were within 8? of the QM regions.After the geometry optimization in QM/MM level, we selected the QM region with H saturation of every stateas the new compound models.Then using the Gaussian 03 program package[29], we employed the time-depen- dent density functional theory (TDDFT) combining with the sum-overstates method[37,38]developed by Cheng’s group[39,40]atthe 6-311++G** basis sets level, to calculate their NLO properties.As the NLO properties only correspond to the static calculation, after geometry optimization in QM/MM level, the influence from molecules outside the QM region can be considered as point charge, which would be induced to the TDDFT calculation to obtain the NLO properties.This above method might be reliableto study the NLO properties of Dreiklang states system in this work.

The compact expression of the tensor component of polarizability and the frequency-dependent first-order hyperpolarizability, which can be obtained from the transition moment, dipole moment and transition energy, can be written as follows:

For the first-order nonlinear response, we are interested in the vector component along the ground state dipole moment direction (vec) and the total hyperpolarizability (tot), which are defined as:

The TPA efficiency can be characterized by the TPA cross-section(), and the latter can be directly related to the imaginary part of second hyperpolarizability(-;,, -) by[41]

where(-;,, -) is the third-order polarizability,denotes the refractive index of the medium,corresponds to the local-field factor and= (2+ 2)/3.Generally,= 1.333 in aqueous and= 1.0 in vacuum.Here,value is set to 1.0 in all calculations for systematic comparison.

We calculated lm(-;,, -) using the sum-over-states (SOS) expression[42, 43].To compare the calculated TPA cross-section value with the experimental value measured in solution, the orientationally averaged (isotropic) value ofis evaluated, which is defined as:

where,=,,

3 RESULTS AND DISCUSSION

3.1 Geometry of different states in Dreiklang

To depict the hydrogen-bonding network among the chromophore and residue Try203, we optimized the geometric structures of four states in Fig.1 by employing the QM/MM method with the QM region including the chromophore, Wat242, residue Try203 and Glu222.And the main geometric parameters are shown in Table 1.The Bnstate has nearly planar chromophore structure with its phenol ring almost parallel to the phenol ring plane of Try203.The similar distances of C(2)–C(5) (4.28 ?) and C(3)–C(4) (4.56 ?) imply the two phenol rings of the chromo- phoric Tyr66 and the neighboring Tyr203 residue are parallel-displace (see Fig.1).And the distance between the centres of the phenol rings from chromophore and Tyr203 is about 3.4 ?.A strong hydrogen-bonding network composed of three H-bonds of O(4)–H(1)···O(1) (2.75 ?), O(1)– H(2)···O(2) (2.76 ?),and O(3)–H(3)···N (3.04 ?) might keep this particular feature of structure and stabilize Wat242 in close vicinity to the C(1) of the chromophore for further light-drive hydration reaction.

Fig.1. QM region of four study states.Bright neutral state and bright anionic state referring to the neutral and anionic chromophore of bright states, respectively; Dark neutral state and dark anionic state referring to the neutral and anionic chromophore of dark states, respectively

Table 1. Selected Optimized Structural Parameters (Bond Lengths (?) and Bond Angles (°)) for the Studied States of Dreiklang

For Ba state, deprotonated chromophore also has a nearly planar structure as that of Bn, and its phenol ring of chromophore develops to a more quinoidal structure than that of Bn.The greatly distinct distances of C(2)–C(5) (4.28 ?) and C(3)–C(4) (4.56 ?) indicate the origin parallel structures of two phenol rings in Bn are broken.This result suggests large structural rearrangement as the proton transfer within the hydrogen-bonding network.As shown in Table 1, a new strong H-bond of O(4)–H(1)···O(5) (2.82 ?) was formed as the H-bond of O(4)–H(1)···O(1)is weakened.The direct H-bond connec- tion between residue Try203 and chromophore causes the phenol ring of Try203 to deviate away from the phenol ring of chromophore and approach to the imidazolinone ring.Further, the phenol ring of Try203 makes a certain angle with the phenol ring of chromophore and breaks the origin parallel structure in Bn.Finally, Try203 also forms hydrogen bonds to Wat242 through a new weak hydrogen bond of O(4)–H(1)···O(1) (3.29 ?).Wat242 can be stabilized again in close vicinity to the C1 of chromophore by new hydrogen bond network which is composed of O(4)–H(1)···O(1), O(1)–H(2)···O(2) (2.79 ?) and O(3)–H(3)···N (3.00 ?).

For the Dark state of Dreiklang, as Wat242 has been used to light-induced hydration of the imidazo- linone ring upon switching from the bright to the dark state, the hydrogen bond network between Try203 and chromophore is broken, and the dis- tances of O(1)–O(4) enlarge to 5.12 and 5.03 ? in neutral and anionic dark states, respectively.However, the phenol ring of Try203 still approaches to the phenol ring of chromophore for both two dark states.The light-induced water split reaction results in the imidazolinone ring of chromophore convert into a 2-hydroxyimidazolidinone ring, and the H(2) transfers to Glu222 with forming the H(2)–O(2) bond, while H(3) transfers to chromophore to generate a H(3)–N bond.This proton transfer and structure adjustment indicate that the proton transfer through Glu222 might be an indispensable process in light-induce water split reaction.Furthermore, depro- tonation of dark chromophore in Da results in the same structure adjustment as that of Dn, except the quinoidal structure of phenol ring of chromophore.The geometry study of different states in Dreiklang clearly illuminates that structures undergo large rearrangement within the light induce switch process.

3.2 Electronic structure and one-photon absorption properties

We employ TDDFT/MM method with B3LYP/6-311++G** level in QM region to calculate the one- photon absorption (OPA) properties of the four states in Fig.1.The protein environment was considered by imposing the point charge from QM/MM geometri- cal optimization results.OPA calculation result is shown in Table 2.And the plots of relevance frontier orbitals of study states are shown in Fig.2.The OPA band of Bn locates at 371 nm with the oscillator strength of 0.5665.This band corresponds tothe second excitation with HOMO-1 to LUMO charac- ters.From Fig.2, both the HOMO-1 and LUMO of Bn completely locate on the chromophore, showingand*characters, respectively.The Ba state has its OPA band at 419.9 nm with oscillator strength of 0.6666.This band corresponds to S2 with HOMO to LUMO characters.As shown in Fig.2, HOMO and LUMO of Ba also completely locate on the chromo- phore, showingand*characters, respectively.Therefore, both the OPA bands of Bn and Ba are mainly contributed by the-conjugation of chromo- phore.

While, these two bands of bright state both overes- timate the excitation energies compared to the regarding experimental measurements of Dreiklang (see Table1).The reason can be attributed to the systematic overestimation of excitation energy in B3LYP calculations, especially for the anionic chro- mophores.Another reason might be the chromophore of QM region in our models more close to a model compound for the chromophore, 4-hydroxybenzy- lidene-2,3-dimethylimidazolinone (HBDI), which is a well-known model compound of the GFP chromo- phore.The OPA bands of HBDI in neutral and anionic states are 370 and 432 nm, respectively.Our calculation bands of two bright states agree well with the regarding OPA experiment data of HBDI.The OPA calculation of Dreiklang illuminates that the-conjugation chromophore mainly dominates the OPA properties of FPs, and the structural integrity of chromophore would sensitively influence the theoretical simulation of OPA properties.

Fig.2. Isosurfaces of the frontier orbitals which participate in the relevant excitations for the studied states

Table 2. One-photon Absorption Properties of Studied States.Oscillator Strengths ?, One-photon Absorption Wavelength λ (nm) and Relevant Excitations

For dark state of Dreiklang, the OPA band of neutral state locates at 328.8 nm with oscillator strength of 0.5407.This band corresponds to the S1 excitation state with HOMO-1 to LUMO characters, and agrees well with the experiment data of 340 nm.From Fig.2, the HOMO-1 and LUMO of Dn state also almost locate on chromophore withto*character mainly locating on the phenol ring of chromophore.Due to hydration reaction, theconjugation of Da’s chromophore is smaller than that of Bn and Ba, which also result in the great blue shift of the OPA band of Dn.Additionally, we also find the strongest OPA band of anionic state located at 388.2 nm, which is neither inconsistent with the experiment data of 340 nm nor deviating from the systematic overestimation of excitation energy in B3LYP calculations.Since the protonated form of chromophore in dark state of Dreiklang has not been identified yet, we suggest that the chromophore of Dreiklang dark state might not be the anionic but neutral.

3.3 The second harmonic generation properties

Before calculating the first-order hyperpolarizabi- litywith truncated sum-over-states (SOS) method, it is necessaryto investigate the behavior of the convergence in the summationof the excited states, in order to obtain reliable results.Fig.3ashows the relationship of the calculated first-order hyperpo- larizability and the number of states for the studied conformers.The results showed that all the first-orderhyperpolarizabilitywas converged before 60 states.Accordingly, all discussionsand studies in the following are based on the truncatedSOS method with 60 excited states.

We obtained the calculated static and dynamic first hyperpolarizabilities by means of TDDFT/MM calculations and SOS method, and the protein environment was considered by point charge field from QM/MM optimization (Table 3).Ba state has the largeststatic first hyperpolarizability of 23.52 × 1030cm5/esu among the three states of Dreiklang.And the Dn state has the smallest static first hyperpolarizability of 9.06 × 1030cm5/esu.We also calculated the dynamic first-order hyperpolarizability(2;,) according to formula (2), in which the laser frequency () is an input parameter in the SOS formulation.For instance, the values of btot at the input energy of 1.165 eV (1064 nm), far from resonance, are 20.59, 68.05 and 14.84 × 1030cm5/esu for the Bn, Ba and Dn states, respectively, with the same order as in the static case.It is interesting that the first-order hyperpolarizabilityamplitude ordering of protonated Bn state and deprotonated Ba state of Dreiklang are quite different from that of Dronpa, another kind of RSPFs, whose protonated chromo- phore form has larger static first hyperpolarizability than the deprotonated chromophore form.Thus, the first-order hyperpolarizability amplitude ordering in Dreiklang might follow with other rules.

Fig.3. (a) Convergent behavior oftotwith the number of excited states; (b) Relationship of the imaginary part of the third-order optical susceptibility and the number of excited state

Table 3. The First-order Hyperpolarizabilities β (× 1030 cm5/esu) of Studied States.Oscillator Strengths?, One-photon Absorption Wavelengthλ (nm) and Relevant Excitations

Significantly, the static first hyperpolarizability values of Bn state (9.53 × 1030cm5/esu) agree well with that of eYFP, which has a centrosymmetric stacking of the chromophoric Tyr66 and the neigh- boring Tyr203 residue.And the static first hyperpolarizability value of Ba state (23.52 × 1030cm5/esu) is consistent well with an eYFP mutant- SHardonnay, which is developed by Meulenaere and coworkers.Through Y203F mutant of eYFP, the original centrosymmetric stacking structure in eYFP has been broken in SHardonnay, so nocentrosym- metric structure changing of SHardonnay greatly improves its first-order hyperpolarizability response.Dreiklang undergoes similar centrosymmetric or nocentrosymmetric structure rearrangement within the Bn to Ba photon switching process.The Dn state of Dreiklang contains the centrosymmetric stacking structure similar to that of eYFP, which causes a substantial drop in the first-order hyperpolarizability of the Dn state, while for the Ba state, the centrosym- metric stacking structure is broken to the nocen- trosymmetric structure as the structural rearrange- ment, which results in the improvement of the first-order hyperpolarizability.So, the first hyperpo- larizabilityproperties of Dreiklang are correlated to the particular geometrical structures of different states.

3.4 Two-photon absorption properties

In this section, we employed the time-dependent density functional theory and sum over state method to calculate the TPA cross section of three confor- mers of Dreiklang.Fig.3b shows the relationships between the imaginary part of the third-order polarizability and the number of states for all conformers studied.It is illustrated that all imaginary parts of the third-order polarizabilities of BCNNRs were converged before 60 states, which ascertains 60 states are enough for the convergence of the third-order optical polarizability.

The selected TPA parameters are listed in Table 4.Our calculation presents that all the three states of Dreiklang did not show the common main TPA band at twice wavelength of their each strongest OPA band.Instead, the TPA spectra of Bn only show a strong band with TPA cross section of 7.99 GM at 608 nm.This band corresponds to its fifth excitation state with HOMO-3 to the LUMO character, while for the Ba state, it has strong band locatinghloyedonverted ties would switch strong or low as the at 596 nm (2.08 eV) with the largest TPA cross section of 15.63 GM among the three states of Dreiklang.This TPA band corresponds to the twelfth excitation with contribution from HOMO-3 to the LUMO character.The anionic chromophore of bright state has the largest TPA cross section.This result is consistent with other GPF-like fluorescence proteins whose deprotonated chromophore form has larger TPA cross section than that of protonated form.The strongest TPA band of Dn locates at 496 nm (2.49 eV) with TPA cross section of 7.98 GM.This band corresponds to a very higher excited state of S21 with contribution from HOMO-1 to the LUMO+6 character, and this band blue shifts above 100 nm compared to other two bright states.For the three states of Dreiklang, their TPA responses are different, because the rearrangement of chromophore structures would change theirconjugation of chromophores which directly affect the TPA of response of the three states of Dreiklang.

Table 4. Two-photon Absorption Properties of STUDIED States.Two-photon Absorption Wavelength λ2 (nm), Oscillator Strength?, Two-photon Absorption Cross Section δ (GM) and Relevant Excitations

Furthermore, we investigate the-stacking interac- tion on TPA properties.We select two snapshots from 2 ns molecular dynamic of Bright neutral states noted as Bn-1 and Bn-2, respectively, whose distances between the centers of the phenol rings from the chromophore and Tyr203 are shorter than that of Bn, about 3.8 and 4.0 ? for Bn-1 and Bn-2, respectively.Their TPA properties are also presented in Table 4.We find that both Bn-1 and Bn-2 have similar TPA cross section value of 10.2 GM, which is larger than that of Bn.The improvement of TPA response mainly attributes to the shorter distance between the centers of phenol rings from chromophore and Tyr203 of Bn-1 and Bn-2, which would enhance the charge transfer among the-stacking systems and further lead to the increase of TPA cross sections.Combined with the preceding paragraph, the TPA properties of Dreiklang are also structural depending.Theconjugation of chromo- phore and the-stacking systems might be two important factors for TPA response enhancement.

4 CONCLUSION

Here we employed the QM/MM method to opti- mize the different states in Dreiklang, and calculate their SHG and TPA properties.The geometrical study of different states in Dreiklang illuminates that the structure undergoes large rearrangement within the light inducing switchable process, which could influence their SHG and TPA properties.Bn state exhibits very low SHG response for its centrosym- metry of-stacking structure, while its TPA responses would be enhanced as two phenol rings get close.The strongest SHG and TPA responses both present in the Ba state of Dreiklang due to its nocentrosymmetric structure and anionic chromo- phore form, respectively.Dark state has similar SHG response as Bn and the lowest TPA response in Dreiklang.In general, the SHG and TPA responses might undergo one changeas the light induces once switchable process among different states.Further- more, we propose for first time that the protonated form of chromophore in Dark state is neutral.This work would prospectively guide the application of Dreiklang on the NLO technology, and help develop new RSFP with special NLO functions.

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15 December 2017;

23 March 2018

①This investigation was based on work supported by the National Natural Science Foundation of China (No.21703246 and 21403242) and Natural Science Foundation of Fujian Province (2014J05021)

.E-mail:chunsen.li@fjirsm.ac.cn

10.14102/j.cnki.0254-5861.2011-1931