Electrostatic Origin of the Red Solvatochromic Shift ... - ACS Publications

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Electrostatic Origin of the Red Solvatochromic Shift of DFHBDI in RNA Spinach Samik Bose, Suman Chakrabarty,* and Debashree Ghosh* Physical and Materials Chemistry Division, CSIR-National Chemical Laboratory, Pune 411008, India S Supporting Information *

ABSTRACT: Interactions with the environment tune the spectral properties of biological chromophores, e.g., fluorescent proteins. Understanding the relative contribution of the various types of noncovalent interactions in the spectral shifts can provide rational design principles toward developing new fluorescent probes. In this work, we investigate the origin of the red shift in the absorption spectra of the difluoro hydroxybenzylidene dimethyl imidazolinone (DFHBDI) chromophore in RNA spinach as compared to the aqueous solution. We systematically decompose the effects of various components of interactions, namely, stacking, hydrogen bonding, and long-range electrostatics, in order to elucidate the relative role of these interactions in the observed spectral behavior. We find that the absorption peak of DFHBDI is red-shifted by ∼0.35 eV in RNA relative to the aqueous solution. Earlier proposals from Huang and co-workers have implicated the stacking interactions between DFHBDI and nucleic acid bases to be the driving force behind the observed red shift. In contrast, our findings reveal that the long-range electrostatic interactions between DFHBDI and negatively charged RNA make the most significant contribution. Moreover, we notice that the opposing electrostatic fields due to the RNA backbone and the polarized water molecules around the RNA give rise to the resultant red shift. Our results emphasize the effect of strong heterogeneity in the various environmental factors that might be competing with each other.

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INTRODUCTION The discovery of green fluorescent protein (GFP) has revolutionized live cell imaging.1−7 Fluorescent proteins (FPs) have been successfully used as biomarkers after the single-point mutations achieved in 1994 by Tsien and coworkers,8,9 which enhanced the fluorescent properties of the protein considerably. Since then, many new fluorescent proteins (FPs) have been engineered by mutating the neighboring amino acids, which lead to a significant shift in absorption and emission maxima of the chromophore.10−17 The origin of these spectral shifts may include structural modifications of the chromophore, such as increased conjugation found in red fluorescent protein (RFP),18 local or specific interactions such as stacking, hydrogen bonding (Hbonding) of the chromophore with adjacent amino acids and waters,12,13,19,20 proton transfer,21−25 and cis−trans isomerization and photoisomerization,25−31 and electrostatic interactions32−40 with the rest of the protein and surrounding water molecules. One such example of a specific-interaction-led spectral variation is mutant Arg96Cys of GFP,17 where the removal of the H-bond between arginine and imidazolinone leads to a blue shift in the absorption (∼15 nm) and emission (∼7 nm) maxima. A recent theoretical study suggests that the Thr62Tyr and Thr203Tyr double mutation in GFP causes a red shift in absorption and emission energies.41 Furthermore, yellow fluorescent proteins (YFP, Thr203Tyr or Thr203Phe) have been engineered as variants of GFP where one threonine residue from the neighborhood of the chromophore (HBDI © XXXX American Chemical Society

anion, i.e., hydroxybenzylidene dimethyl imidazolinone) is replaced by an aromatic amino acid (tyrosine or phenylalanine), causing a red shift (∼20 nm) in both the absorption and emission maxima.17,19 This red shift has been proposed to be driven by the stacking interactions between the phenolate ring of the HBDI chromophore and the phenol or benzene moieties of tyrosine or phenylalanine, respectively.13,42,43 However, Kummer and co-workers have reported a red shift of ∼10 nm in the aliphatic mutations of Thr203, e.g., Thr203Ile, which indicates that the removal of Thr203-HBDI hydrogen bonding in YFP variants plays a significant role in the spectral shift as well.12,17 Therefore, it is evident that one can achieve desirable photophysical applications by tweaking the noncovalent interactions between the chromophore and its environment. There is a continuous effort toward understanding and tuning these interactions, which has led to the successful engineering of the FPs over the years.12,13,17,41,44−49 Although biomarkers for proteins have been successfully achieved with the help of FPs, tracking RNA molecules in vivo has always been a challenging problem owing to the lack of intrinsic fluorescence in most of the nucleic acid base sequences.50−52 Recently, remarkable success has been achieved in extending the repertoire of GFP chromophores to RNA.53−55 A particular type of double-stranded RNA environReceived: March 15, 2017 Revised: April 24, 2017 Published: April 24, 2017 A

DOI: 10.1021/acs.jpcb.7b02445 J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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COMPUTATIONAL DETAILS The X-ray structure of RNA spinach (PDB ID: 4KZD) was used for our computational study of the DFHBDI chromophore in G-quadruplex-rich RNA. Nucleic acid bases (NABs) beyond 25 Å from the chromophore were not considered for our simulation and vertical excitation energy (VEE) calculations. The detailed rationale for choosing a small subsystem is explained in the SI (sections 1 and 2). In the MD simulation of the chromophore and RNA system, we considered a dodecahedral box with 26880 water molecules and 29 sodium ions to neutralize the system. AMBER03 and TIP4P-Ew force fields were used for RNA and water, respectively,60,61 whereas the force field parameters for the DFHBDI anion were generated by the combination RED server facility using restrained electrostatic potential (RESP) fit charges and the ANTECHAMBER package.62−64 A classical force field energy minimization of the system was carried out, constraining the heavy atoms of RNA and the chromophore in the Gromacs 5.0.5 package,65 followed by QM/MM energy minimization using Gromacs (5.0.5) and the ORCA (3.0.3)66 interface constraining only the heavy atoms of RNA, with B3LYP/6-31G as the QM level of theory. The QM/MM optimized geometry was used for further analysis of the competing factors toward the solvatochromic shift of the DFHBDI chromophore. Furthermore, classical NVT equilibration was performed at 300 K for 100 ps constraining the heavy atoms of RNA and the chromophore followed by a QM/MM NVT simulation at 300 K for 50 ps without any constraint on the system. Equally spaced (1 ps) snapshots from the QM/MM NVT simulation were used to understand the dynamic behavior of the system. The deprotonated structure of DFHBDI was optimized at the ωB97X-D/6-31+G* level of theory using the Q-Chem 4.2 package.67 Benchmark VEEs of HBDI and DFHBDI anions were calculated with the equation-of-motion excitation-energy coupled-cluster (EOM-EE-CCSD) and spin opposite scaled configuration interaction with perturbative doubles (SOSCIS(D)) methods. The calculations were performed at the EOM-EE-CCSD/6-31+G*, EOM-EE-CCSD/6-311++G*, CAM-B3LYP/6-31+G*, CAM-B3LYP/6-311++G*, SOS-CIS(D)/6-31+G*, SOS-CIS(D)/6-311++G*, and SOS-CIS(D)/631+G*(heavy atoms)-3-21G(for hydrogen) levels of theory. The mixed 6-31+G*(heavy atoms)-3-21G(hydrogens) basis is denoted as a mixed basis in this article. SOS-CIS(D), as a QM method for FP chromophores, has been shown to produce reliable excitation energies previously and has been used extensively for HBDI and DFHBDI chromophores.35,59,68,69 Subsequent VEE calculations were made in the SOS-CIS(D)/ mixed basis level of theory. The mixed basis is benchmarked against other basis sets in the SI (Section 3, Table S2). VEEs of all of the subsystems, such as DF+4NAB(stacked), DF +8NAB(stacked), DF+Sugar, or DF+Guanine (stacked model), are also computed for comparison. QM/MM calculations with DFHBDI in the QM region and the rest of the system in the MM region were performed at the same level of QM theory and MM charges from the force fields of the MD simulation. Additionally, to understand the effect of the environment on the excitation energy we computed the field produced by each component of the environment as well as the whole biological environment over the bridge carbon of the chromophore for 1000 equally spaced snapshots from the QM/MM MD simulation. A detailed description of the field computation

ment in spinach aptamer has been reported to enhance the binding of the GFP chromophore variant (DFHBDI anion, i.e., difluoro hydroxybenzylidene dimethyl imidazolinone) and activate its fluorescence.54 The wide range of biological applications with this RNA analogue of GFP has already been discussed in the literature.55−58 It has been proposed that the major stabilizing factor for the DFHBDI-RNA complex (Figure 1) is the stacking interaction of DFHBDI with the adjacent

Figure 1. Crystal structure of a DFHBDI chromophore bound to RNA spinach (PDB ID: 4KZD). The local environment of DFHBDI is shown in the inset highlighting the nearest nucleic acid bases (NABs).

guanines in the G-quadruplex region, e.g., 23G and 54G, as well as H-bonding interactions with the planar guanine (28G).54 Compared to an aqueous solution, a red shift of ∼40 nm (∼0.30 eV) has been reported in the absorption maxima of the DFHBDI chromophore in RNA (biological medium).53 Analogous to YFP, it has been primarily attributed to the stacking interactions between DFHBDI and the guanines in the G-quadruplex region.54 However, the origin of the solvatochromic shifts in the absorption and emission spectra of the chromophore as well as the environment−chromophore interaction inside GFP and RNA is expected to be different because these environments are significantly different. Although a recent computational report discusses the absorption and emission energies of the system,59 there exists no systematic investigation of the relative contributions of various types of chromophore−environment interactions, namely, stacking and electrostatics toward the observed red shift in the absorption spectra. The objective of the current work is to (i) reproduce the spectral shift in the absorption maxima of DFHBDI in the RNA environment, (ii) elucidate the local/specific interactions (stacking and H-bonding) and the nonspecific long-range electrostatic interactions between the chromophore and its environment, and (iii) understand the relative contribution of these various types of interactions toward the observed solvatochromic shift in the absorption maxima. In this work, we have carried out hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations as well as single-point QM/MM and full QM excited-state calculations to understand the static and dynamic contributions of the chromophore−environment interactions toward the absorption spectra of DFHBDI in spinach RNA. We have dissected the various types of chromophore−environment interactions systematically to elucidate their relative role in the observed solvatochromic shift in this system. B


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stacking in VEE with a model stacked DFHBDI anion−guanine system. It should be noted that among the three different types of NABs that are in close proximity to DFHBDI (with possible stacking interactions), guanine has the lowest VEE and hence is chosen as the constituent to model the stacked system. Figure 2

from the MD simulation snapshots is provided in the SI (Section 1). All of the structures that were used for the calculation of VEE are also given in the SI.

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RESULTS AND DISCUSSION The interaction between a chromophore and its environment can be broadly divided into long-range and short-range interactions. Long-range interactions are electrostatic in nature because the distance-dependent decay is slowest with respect to electrostatic (charge−charge) interactions ( 1 decay). On the R other hand, short-range interactions can be stacking-induced or specific electrostatic interactions, such as H-bonding. We have therefore decomposed the spectral shifts due to each of these interactions. Here, it should be noted that there can be secondary effects due to stacking such as structural rigidity caused by the nearby stacked NABs. Thus, we have also studied this indirect effect of structural rigidity on the spectral shift. Polarization is an important component of electrostatics along with Coulomb interactions (effect of static multipoles or charges). With regard to GFP, it has been shown that polarization is important to predicting the qualitative nature (red or blue shift) of the solvatochromic shift.37,70 One general approach to including all of the electrostatic and polarization effects is to increase the QM subsystem (cluster size) to include all interactions.33,70 Such a technique, although unsustainable for large cluster sizes (to include long-range electrostatics), can be used to understand local or specific interactions. We have therefore used such large QM (cluster size) subsystem studies to evaluate the effect of stacking and immediate H-bonds with the chromophore. Furthermore, it has been found that polarization plays a relatively small role (∼0.03 eV) in the solvatochromic shift of the DFHBDI anion in aqueous solvation.71 We have been able to predict the correct solvatochromic shift (blue shift) of the DFHBDI chromophores in water using QM/EFP (effective fragment potential) with and without polarization.71 These factors motivate us to use the simple MD-QM/MM formalism to understand the effect of long-range electrostatics in the spectral shift of DFHBDI in the RNA spinach system. Moreover, the gas-phase VEE obtained by using the SOSCIS(D) method (2.55−2.58 eV) for the DFHBDI anion is in good agreement with the VEE of the same anion reported in ref 59 using second-order perturbation-corrected multistate complete active space (MS-CASPT2) and symmetry-adapted cluster configuration interaction (SAC-CI) methods (2.46− 2.60 eV). Also, our estimated VEE of the DFHBDI anion at the CAM-B3LYP/6-311++G* level of theory (3.09 eV) is in excellent agreement with the VEE (3.11 eV) computed at the CAM-B3LYP/6-311+G** level of theory in ref 59. Role of Stacking Interactions. Figure 1 shows the X-ray structure of the DFHBDI chromophore in RNA spinach, and the inset shows the chromophore (DFHBDI anion) and the nearest NABs. The π-stacking interactions with 23G and 54G have been proposed to be responsible for the red shift of absorption spectra as well as the small Stokes’ shift of DFHBDI in RNA.54 To test this hypothesis, here we explore the effect of interactions of DFHBDI with the nearest NABs based on the available crystal structure. The reduction in the HOMO−LUMO energy gap (π−π* vertical excitation energy) can provide a direct signature of the presence of stacking interactions. We investigate the effect of

Figure 2. Effects on HOMO and LUMO due to stacking interactions between (i) the DFHBDI anion with guanine in a model stacked geometry and (ii) DFHBDI with four nearest NABs from the RNA crystal structure (QM/MM-optimized). DF and Gua refer to DFHBDI and guanine, respectively.

indicates a red shift in the π−π* vertical excitation of this model system in comparison to that of the DFHBDI anion, with an increase in the energy of HOMO and a decrease in the LUMO energy. To ascertain whether such a stackingdependent red shift is present in the absorption spectra of DFHBDI in RNA spinach, we have calculated the VEE of DFHBDI with the four nearest stacked NABs (23G, 50U, 53A, and 54G) in its QM/MM optimized geometry. The SOSCIS(D)/mixed basis was used for the VEE calculations. Apart from the model DFHBDI anion−guanine system, Figure 2 also compares the VEE of DFHBDI in the gas phase and DFHBDI +4NABs (in the QM/MM optimized crystal structure). Surprisingly, the VEE of the DFHBDI+4NABs system shows a slight blue shift (∼0.04 eV) with respect to DFHBDI in vacuum! The lack of orbital overlap between DFHBDI and the NABs indicates that the effects of stacking are minimal. It should be noted that the stacking-induced red shift is strongly dependent on the distance and orientation and rapidly decreases with distance because it is dependent on the orbital overlaps and dispersion interactions (which has a 1/R6 dependence). While estimating the effect of stacking via the domino effect due to the next nearest neighbors (i.e., DFHBDI +8 stacked NABs as shown in Figure S3 in SI), we observe that the contribution of stacking due to this second layer of NABs in the VEE of DFHBDI is insignificant (blue shift of ∼0.01 eV). The small blue shift due to the nearest neighbor stacked NABs on the DFHBDI chromophore is also observed along the QM/MM MD trajectory (Figure 3). The VEEs of 10 equally spaced DFHBDI+4NAB subsystem snapshots taken from the MD trajectory (calculated with a full SOS-CIS(D)/mixed basis level of theory) show that there is an average blue shift with respect to the gas-phase chromophore along the trajectory. Thus, we conclude that the interaction between nearestneighbor stacked NABs cannot be the reason behind the red C


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Table 1. ΔVEE of the DFHBDI Anion Due to Its Interaction with the Nearest-Neighbor Nucleosides subsystems

ΔVEE (eV)

DF+Sugar(23G-stacked) DF+Sugar(50U-stacked) DF+Sugar(53A-stacked) DF+28G(planar guanine)

+0.07 +0.01 −0.01 −0.08

analogous to the HBDI anion,72,73 a significant amount of electronic charge is transferred from the phenolate ring to the bridge and imidazolinone ring during electronic excitation, thus reducing the 23G sugar−phenolate oxygen(DFHBDI) Hbonding interaction in the excited state. Hence, H-bond donation by the 23G sugar at the phenolate oxygen of the DFHBDI anion preferentially stabilizes the ground state, giving rise to a blue solvatochromic shift in VEE. A similar trend in the blue shift is expected due to the Hbond donation in 28G-DFHBDI (Figure 4a). However, our calculations reveal a significant red shift (∼0.08 eV) caused by this planar guanine. Interestingly, an Arg96Cys variant of GFP that lacks H-bond donation at the carbonyl O of the HBDI chromophore causes a blue shift in the absorption maxima.17,44 Subsequently, it was proposed by Tsien that the removal of the H-bond at the carbonyl O leads to this blue shift in the absorption spectra.17 This indicates the possibility that H-bond donation at the imidazolinone CO produces a red shift in the VEE of the chromophore.20 The reason behind this red shift is the charge transfer from the phenolate ring toward the benzylidene C on the bridge and the imidazolinone of the HBDI anion during the excitation, leading to stronger Hbonding interactions in the excited state, thus stabilizing the excited state and lowering the VEE. NBO analysis (SI Table S4) and the attachment−detachment electron density of the DFHBDI anion (Figure 5) clearly indicate that the carbonyl O of the imidazolinone ring gains electron density in the excited state. Hence, H-bond donor NH and NH2 groups of 28G preferentially stabilize the excited state of the DFHBDI anion, resulting in the red shift in VEE of DFHBDI. H-bond donation via water at imidazolinone CO also reproduces this red shift (SI, Table S6). The other electrostatic interaction of DFHBDI-28G is between the carbonyl oxygen of

Figure 3. Blue shift in VEE of the DFHBDI+4NAB systems in 10 equally spaced configurations obtained from the QM/MM MD trajectory.

shift in the absorption spectra of DFHBDI in RNA spinach due to poor orbital overlap. Role of H-Bonds and Specific Electrostatic Interactions. Apart from the stacking interactions with the nearest NABs, DFHBDI is also stabilized by H-bonds and electrostatic interactions with the planar guanine (28G) and sugar moieties of the nearest nucleotides (Figure 4a and SI Figure S4). The distances between DFHBDI and the nearest neighbors in the QM/MM-optimized configuration are compared to the distances reported by Li and co-workers.59 As seen from the SI (section 10) and Figure 4b, QM/MM-optimized distances are in good agreement with the reported distances. The SOS-CIS(D)/mixed basis calculations with QM/MMoptimized geometry indicate that the A53 (sugar)-DFHBDI and U50 (sugar)-DFHBDI H-bonds have negligible effects on the spectral shift of DFHBDI (Table 1). However, 23G (through sugar) and planar 28G (through guanine) (shown in Figure 4a) have H-bond and polar interactions with DFHBDI resulting in a significant shift in its VEE. The interaction of 23G with DFHBDI causes a blue shift of ∼0.07 eV. This is attributed to the H-bond donation by the sugar of 23G to the phenolate oxygen of DFHBDI. Natural bond orbital (NBO) charge analysis of ground and excited states of DFHBDI anion (SI, Table S4 and S5) shows that,

Figure 4. (a) Specific electrostatic interactions and H-bonding with the nearest NABs and sugars in the QM/MM-optimized geometry and MD simulation trajectory. (b) The distribution of O···H distances between the DFHBDI chromophore and nearby nucleosides is shown. Specific pairs of O···H atoms are marked a−d, as above. D


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noted here that the average O···H distances from the QM/MM MD trajectory are in good agreement with the previously reported O···H distance of DFHBDI in RNA spinach.59 In summary, among the specific interactions between the chromophore and environment, the electrostatics rather than the stacking interactions are the key to the red shift in the absorption spectra of DFHBDI in RNA. Role of Long-Range Electrostatic Interactions. Apart from short-range specific interactions between the neighboring NABs and the DFHBDI moiety, long-range electrostatic interactions with the negatively charged RNA and surrounding water + ions can affect the spectral behavior. Figure 6a shows the spectral shift in the VEE of DFHBDI due to different components of the system (RNA and water + ions) at the QM/ MM-optimized geometry. We notice an overall red shift of 0.11 eV in the VEE of DFHBDI due to the environment. A subsystem such as RNA−environment (without water and ions) gives rise to a red shift of 0.17 eV, whereas the aqueous environment (water + ion) gives rise to a blue shift of 0.15 eV. It is important to note that in case of the HBDI anion it is experimentally reported that the aqueous solution causes a blue shift. Furthermore, it has been noticed that the polarity and proticity of the solvent medium have significant effects on the solvatochromic shift, which is indicative of a significant chargetransfer nature of the excited state.44,74 In our previous studies, we have shown that hybrid QM/EFP can predict this blue shift accurately and the effect of polarization itself is quite small in pure water.71 Now, comparing the effect of water in a biological system to that of the pure water environment on DFHBDI71 (0.10 eV blue shift) indicates that the water around RNA is more directional in nature and tries to oppose the effect (red shift) due to the RNA backbone. Interestingly, our results also indicate the short-range stacking interaction, which causes a slight blue shift (∼0.04 eV) in the VEE of DFHBDI to be overridden by the long-range electrostatic interaction of RNA. Moreover, the qualitative trend of the spectral shifts that we have observed in the QM/MM-optimized geometry holds good along the MD trajectory. A comparison of Figures 6a (QM/ MM minima) and 7 (QM/MM MD trajectory) shows that the spectral shifts due to RNA and the surrounding water and ions are in opposite directions. Figure 6b shows the simulated absorption spectra of DFHBDI in the biological media computed by QM/MM calculations using 50 equally spaced snapshots from the QM/ MM MD trajectory. It predicts an average red shift of 0.25 eV in the absorption maxima with respect to the gas-phase

Figure 5. Pictorial representation of the attachment and detachment of electronic charge (qi,ES − qi,GS), where qi,ES and qi,GS refer to the NBO charges in the excited and ground states, respectively. The ground- and excited-state charges are calculated at the CCSD/6-31G* and EOMCCSD/6-31G* levels of theory. Red and black circles denote the attachment and detachment of electronic charges, respectively. The radius of the circles is proportional to the magnitude.

guanine (28G) and the H attached to the benzylidene C on the bridge of DFHBDI. From the attachment−detachment density (Figure 5) and NBO analysis (Table D in SI), it is also observed that the bridge C gains significant charge density in the excited state, thus giving rise to a large dipole on the bridge CH bond in the excited state. Hence, electron-donating atoms around the hydrogen of the CH bond would preferentially stabilize this dipole via short-range electrostatics and therefore the excited state. The carbonyl oxygen of 28G (Figure 4a), therefore, causes a further red shift in the VEE of the DFHBDI anion. In summary, the red-shifted VEE of 28G-DFHBDI is attributed to the preferential excited-state stabilization by both 28G-NH and NH2 H-bonds with the carbonyl oxygen (DFHBDI) and the short-range electrostatic interaction of the carbonyl oxygen (28G) with the dipole across the CH bond of the DFHBDI anion. To ascertain the importance of these interactions, a histogram of relevant distances along the QM/MM MD trajectory, is plotted in Figure 4b. We observe that the average distance between sugar H(23G) and phenolate O(DFHBDI) increases considerably under thermal fluctuation. However, the different types of specific electrostatic interactions between 28G and DFHBDI remain intact throughout the trajectory. This leads us to conclude that among the short-range specific interactions it is 28G planar to DFHBDI that plays the most significant role in the solvatochromic shift of the chromophore in RNA. This is in accordance with the previously reported experimental and theoretical observations. It should also be

Figure 6. (a) Spectral shift due to the various components of the environment in the QM/MM-optimized geometry. DF refers to the DFHBDI chromophore. (b) Overall simulated spectra of DFHBDI in RNA calculated over the entire MD trajectory using 50 equally spaced configurations. E


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DFHBDI spectra and 0.35 eV with respect to the spectra in the aqueous solution, in reasonable agreement with the experimental observations.54 Furthermore, it should be noted that our average estimation of the spectral shift lacks the contribution of polarization as a result of using an electronicembedding-based QM/MM scheme. Because of our previous observation in aqueous solution that polarization plays a minimal role (0.03 eV) and also the fact that we can reproduce the overall red shift of DFHBDI in RNA, we neglect the effect of polarization in this study. At this point it should be noted that a recent similar QM/ MM study59 has benchamrked the absorption and emission energies of DFHBDI in spinach against the available experimental results. The VEE of DFHBDI in spinach reported here (2.30−2.35 eV) is in good agreement with the VEE calculated at the SAC-CI/D95(d) and LT-DF-LCC2/cc-pVDZ levels of theory in the previous work (2.26−2.45 eV). However, the estimated VEE using MS-CASPT2/6-31G(d) is 0.20−0.35 eV higher than our predicted VEE of DFHBDI in spinach. Figure 7 shows the component-wise VEEs and the electrostatic field experienced by DFHBDI along the bridging C atom. (See the SI for details of the field calculation.) We have also included the VEEs and field due to pure water as a

Figure 7. Evidence of coupling between the electrostatic field exerted by the environment on the DFHBDI chromophore and the observed VEE. Each data point on the plot correspond to a snapshot taken from the MD simulation trajectory. The green and blue points refer to the VEEs as calculated due to only the RNA backbone and water + ions, respectively. The red points refer to that due to the complete environment (RNA, water, and ions), and the pink points are those for pure water.

Figure 8. Effect of structural variation due to the RNA environment. (a) Dihedral angles that are used as a measure of the structural variation are shown. (b) Distribution of the torsional angle [abs(180° − τ(1))] calculated from the MD trajectories in RNA and water, respectively. (c) Effect of the change in dihedral angle on the VEEs. The histogram shows the distribution of the VEEs of the DFHBDI chromophore only, where configurations are taken from the MD trajectory. F


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The Journal of Physical Chemistry B reference system. We notice that the fields produced by RNA and water + ions are in two opposing directions, as is the shift in VEE due to those components. The field produced by the full biological system (RNA + water + ions) is dominated by the field due to RNA and therefore gives rise to the resultant red shift in the spectra. Furthermore, it is the sugar−phosphate backbone of the RNA that has the predominant effect on the red shift and not the stacked NABs (see SI, Figure S6). Role of Structural Variations in Chromophores. There are further secondary effects of the environment, especially those of the change in possible configurations due to the presence of stacked NABs. Therefore, the DFHBDI anion in the RNA environment has different geometry from that of the gas-phase-optimized DFHBDI anion. This in turn has an effect on the spectral shift. The major difference between gas-phaseoptimized and condensed-phase average geometries occurs in the two dihedral angles between the phenolate and imidazolinone rings, as shown in Figure 8a and denoted by τ(1) and τ(2). In contrast to its gas-phase planar geometry (τ(1) = τ(2) = 180°), the dihedral angles between the two rings in RNA vary between 160 and 200°. (Figure 8b shows [abs(180° − τ(1))], which varies between 0 and 20°.) However, this variation is less than what was earlier observed in an aqueous solution of DFHBDI anions (130 to 230°; as shown in Figure 8b, [abs(180° − τ(1))] varies between 0 and 50°).71 This reduced degree of freedom can be attributed to the combined stacking and H-bonding interactions of the chromophore and the RNA environment, which are absent in aqueous solution. We have calculated the VEE of the DFHBDI anion in 20 equally spaced snapshots from the MD simulation trajectory and compared it against the gas-phase VEE. Figure 8c shows that the DFHBDI anion in a biological system has an average −0.10 eV shift (red shift) due to the structural variations. This can be compared to the spectral shift of −0.13 eV due to structural variations in an aqueous environment. The relative insensitivity of the spectral shift (−0.10 vs −0.13 eV) with respect to the structural effects due to the significantly different environment (RNA vs water) is surprising. It shows that the secondary (structural) effects of stacking on the red shift are minimal.

medium. Surprisingly, the phosphate and sugar moieties of the RNA backbone give rise to larger red shifts than in the NABs. Our observations clearly demonstrate that electrostatics plays a more significant role, in both short and long-range interactions, than the stacking interactions (dispersion) in the solvatochromic shifts of the DFHBDI chromophore in RNA spinach. This important finding provides a new direction toward chromophore engineering. However, it is important to note that stacking might have some secondary effect toward constraining the motions of the chromophore, thereby increasing the fluorescence efficiency. Previous studies on YFP had shown that both stacking and electrostatics were almost equally important with respect to the red shift in the fluorescence spectra.42 Our observation of the greater importance of electrostatics in the spinach aptamer, which is a distinctly different environment than the YFP, shows that the predominant role of electrostatics might be a more fundamental and universal aspect.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b02445. Computational details (including field calculation), benchmark QM calculations, convergence of field with distance from chromophore, effects of perfect stacking, effect of long-range stacking, effect of stacking along the MD trajectory, effect of H-bonding with neighboring nucleosides, NBO charges of the chromophore in the ground and excited states along with attachment− detachment densities and geometries of the QM systems used in various calculations (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +91 20 2590 3053. *E-mail: [email protected]. Phone: +91 20 2590 3052. ORCID

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Samik Bose: 0000-0002-0273-9162 Suman Chakrabarty: 0000-0002-9461-0015

CONCLUSIONS From the MD simulation and the QM/MM-optimized geometry, we notice that the effect of stacking interactions of DFHBDI with nearest-neighbor NABs on the spectral shift of DFHBDI is minimal (blue shift). This is contrary to previous proposals of the spectral tuning of the HBDI anion41 and of YFP-like stacking interactions in RNA spinach and is due to the unfavorable distance and orientation between the DFHBDI and NABs. Furthermore, H-bonding interactions with the nearestneighbor nucleosides can lead to both varying magnitude as well as the direction of spectral shifts depending on whether the H-bonding is through the phenolate O or the carbonyl O atom of the chromophore. Among specific electrostatic interactions, the H-bonds between 28G-NH or NH2 with carbonyl O of DFHBDI and the interaction of 28G-CO with benzylidene H (bridge) of DFHBDI cause the maximum red shift and are persistent throughout the MD trajectory. Long-range electrostatic interactions are crucial to the observed red shift in the absorption spectra and are governed by the opposing effects of the negatively charged RNA backbone and the screening effect of the surrounding aqueous

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the CSIR XIIth Five Year Plan on Multiscale Modelling for funding and CSIR-4PI for computational resources. D.G. thanks DST-SERB and DAE-BRNS for additional funding. S.C. is thankful for a DST-SERB Ramanujan Fellowship. S.B. thanks CSIR for a Senior Research Fellowship and the Ph.D. program of AcSIR. D.G. thanks Prof. Anna I. Krylov for many insightful discussions and also valuable comments on the article.

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