Molecular Dynamic Indicators of the Photoswitching Properties of

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Molecular Dynamic Indicators of the Photoswitching Properties of GFPs Daryna Smyrnova, Benjamien Moeyaert, Servaas Michielssens, Johan Hofkens, Peter Dedecker, and Arnout Ceulemans J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b04826 • Publication Date (Web): 25 Aug 2015 Downloaded from http://pubs.acs.org on August 31, 2015

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The Journal of Physical Chemistry

Molecular Dynamic Indicators of the Photoswitching Properties of GFPs Daryna Smyrnova,† Benjamien Moeyaert,‡ Servaas Michielssens,† Johan Hofkens,‡ Peter Dedecker,‡ and Arnout Ceulemans∗,† Quantum Chemistry and Physical Chemistry Division, and Molecular Imaging and Photonics Division, Department of Chemistry, KU Leuven, Celestijnenenlaan 200F, 3001 Heverlee, Belgium E-mail: [email protected]

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To whom correspondence should be addressed Quantum Chemistry and Physical Chemistry Division, Department of Chemistry , KU Leuven, Celestijnenenlaan 200F, 3001 Heverlee, Belgium ‡ Molecular Imaging and Photonics Division, Department of Chemistry, KU Leuven, Celestijnenenlaan 200F, 3001 Heverlee, Belgium †

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Abstract Reversibly photoswitchable fluorescent proteins (RSFPs) are highly useful probes for a range of applications including diffraction-unlimited fluorescence microscopy. It was previously shown that reversible photoswitching involves not only cis-trans isomerization and protonation-deprotonation of the chromophore, but also results in a marked difference in β-barrel flexibility. In this work, we performed flexibility profiling and functional mode analysis (FMA) using molecular dynamics calculations to study how the flexibility of the RSFP β-barrel influences the photoswitching properties of several fluorescent proteins. We also used Partial Least-Squared (PLS) FMA to detect promising mutation sites for the modulation of photoswitching properties of RSFPs. Our results show that the flexibility of RSFP does depend on its state with a systematically higher flexibility in the darkstate compared to the bright-state. In particular our method highlights the importance of Val157 in Dronpa, which upon mutation yields striking difference in the collective motions of the two mutants. Overall, we show that PLS-FMA yields information, complementary to static structures, that can guide rational design of fluorescent proteins.

Keywords RSFP, PLS, FMA

Introduction Fluorescent proteins (FPs) are 30kDa proteins with a β-barrel structure. After translation and folding, they autocatalytically form a chromophore, requiring only the presence of molecular oxygen. 1,2 This unique feature has made them particularly useful for in vivo fluorescence imaging. Their biophysical and spectroscopic properties have been modulated by mutating the amino acid sequence, resulting in brighter and more photostable fluorescent proteins, ranging over almost the entire visible spectrum. 3–9 2


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An interesting class of FPs is the photoactivatable FPs (PAFPs). These labels can change their emissive properties on demand. For instance, reversibly photoswitchable FPs (RSFPs) such as Dronpa 10–12 are originally in a bright state, and can be repeatedly turned off with blue and back on with purple light. Other PAFPs such as the irreversibly photoconvertible FPs (PCFPs) originally exist in a bright green-emissive state, and can by application of a high flux of purple light be irreversibly converted to a red-emissive state. Examples of PCFPs are EosFP 13 and Dendra2. 14 Some recently developed proteins combine both reversible photoswitching and irreversible photoconversion, such as IrisFP, 15 NijiFP 16 and pcDronpa. 17 The interest in PAFPs is mainly driven by the development of diffraction-unlimited fluorescence microscopy techniques like PALM, RESOLFT and pcSOFI 18–24 which all heavily rely on photoswitching or photoconversion of FPs. With these superresolution techniques, an arbitrarily high resolution can in principle be achieved, although the results depend on both instrumental parameters and the properties of the labels that are used. 25 Ideally, RSFPs have two thermostable states that are spectrally well separated, have a high resistance to photobleaching and reliable switching rates. 26,27 The molecular mechanism underlying photoswitching has been the subject of intense study. In most cases the absorption band of the bright fluorescent form of RSFPs is consistent with the deprotonated, anionic state of the chromophore, while the dark, non-fluorescent state corresponds to the neutral form of the chromophore. From crystal structures of RSFPs in both states, it is also seen that the bright state chromophore is in a cis-conformation, while the dark state assumes a trans conformation due to a so-called hula-twist around the chromophore’s methylene bridge 12,28–34 or a rotation around a double bond 35–38 (see Fig. 1). Additionally, NMR studies of the bright and dark states of Dronpa suggest a different flexibility of these two states. 39 In this work we have investigated whether this difference in flexibility observed in Dronpa is a recurring theme in other switchable proteins, by investigating this effect in pairs of

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closely-related switchable proteins that differ by only a few mutations yet exhibit different switching properties. We also have analyzed whether this could serve as a simple indicator of the photoswitching ability of the protein.

Figure 1: Structural and photophysical characteristics of Dronpa. (A) Photoswitching scheme of the Dronpa protein. Upon irradiation with 405 and 488 nm light, Dronpa switches between dark and bright states. (B) Representative views of the chromophore and its nearby residues. Structures of the dark and bright state are shown in gray [Protein Data Bank (PDB) ID code 2POX] and green (PDB ID code 2Z1O), respectively. 12,40 The CYG chromophore in Dronpa adapts distinct cis and trans conformations in the bright and dark state, respectively. (C) Numbering of the β-sheets in barrel-fold of the FP. In particular, we studied two sets of closely related proteins with different switching properties. First, we studied Dronpa and its mutants rsFastLime, 12 rsKame 41 and pcDronpa. 17 Although differing in only 1 to 5 residues, these proteins have markedly different photoswitching properties (see Table 1). In particular rsKame is slow-switching (has lower photoisomerization quantum yield in comparison to Dronpa) and rsFastLime is fast-switching (has 4


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higher photo-isomerization quantum yield in comparison to Dronpa). In the case of pcDronpa only the photoswitching green species was considered for the study. Photo-isomerization quantum yields for all of the proteins presented in this study are summarized in the Table S1. We additionally studied EosFP and IrisFP (like in the case of pcDronpa we considered only the green photoswitchable species before the irreversible green-to-red photoconversion). These two proteins have a 76% sequence similarity compared to Dronpa and differ by only two residues (IrisFP = EosFP_F173S_F191L). The 191 residue is pointing out of the βbarrel and the mutation at this position is said to have no influence on the spectroscopy. 15 However, while EosFP is considered to be non-photoswitchable, IrisFP does photoswitch; its photoswitching properties are attributed to a higher spatial freedom of the chromophore in its pocket and the disruption of a Van der Waals connection between Met159 and hydroxybenzilidene moiety. 15,42 Moreover, the HYG chromophore of EosFP/IrisFP, pivotal for the green-to-red photoconversion property, can also be found in pcDronpa. Here, we examine the β-barrel flexibility of the dark and bright state of Dronpa and some of its slower and faster switching variants. By means of Functional Mode Analysis (FMA) we elucidate the positions of the aminoacids contributing the most to the difference in flexibility between the on- and off-state and then see how this contribution changes in the closest mutants. Table 1: Effect of several mutations on the photoswitching properties of Dronpa. Key mutation(s) V157G V157I V60A+C62H+N94S+N102I+E218G

Mutant rsFastLime 12 rsKame 41 pcDronpa 17

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Properties Fast photoswitching Slow photoswitching Slow photoswitching,green-to-red photoconvertible



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Methods Chromophore parametrization For the parametrization of the chromophores in the cis-state, the input structures were taken from the PDB (Dronpa bright state: 2Z1O and IrisFP bright state: 2VVH). The trans-state structures were created manually using Avogadro. 43 This was done by rotating the hydroxyphenyl moiety of the chromophore around the chromophore’s methylene bridge. Further each chromophore was optimized in vacuum by high level QM method. Also each chromophore was capped by ACE (acetylated) and NME (methylamidated) residues following the standard charge derivation procedure for the amberFF9X. 44 For structure preparation, the parameters were determined from quantum chemical calculations at HF/6-31G* level using Gaussian09. 45 The ESP charges were determined with the R.E.D. tool, 46 which aided in automatizing the optimization of the structures and later on to map the atomic charges. The dihedral angle parameters for φ and θ were taken from literature 29 where they were on the B3LYP/6-31G* level. Parameter files obtained with AmberTools 47 were converted to Gromacs format using the Acepype script. 48 The structures of the chromophore and sidechains with the atomtypes and the atomic point charges can be found in Fig. S1.

Molecular dynamics simulations For the first part of the study, proteins in both the bright and dark state were taken from the PDB (bright structures as mentioned in the previous paragraph, Dronpa dark state: 2POX, IrisFP dark state: 2VVJ, pcDronpa green-on state: 4HQ8, pcDronpa green-off state: 4HQ9, EosFP green state: 1ZUX). Taking into account the importance of the crystallographic waters we have carefully examined the PDB files used in the simulation. In particular for Dronpa several of them are available. The X-ray structure with the highest resolution is 2Z1O, also we compared it to 2Z6X, 39 2IOV, 12 2IE2 49 structures and found that it includes all of the important crystallographic waters present in the other structures. It looks like the 6


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2IOV chain A structure contains an extra water inside of the chromophore’s cavity, however this water is absent in chains B,C and D, hence we have mapped it as of lesser importance and avoided including it to our simulations. Comparison of these four structures (only chain A) can be found in Fig. S2. In order to examine transient states, we replaced, in the bright state structure, the bright state chromophore by the dark state chromophore and changed the protonation of His193 in the bright state’s crystal structure. All of the mutations were made using the Rosetta package. 50 MD simulations were performed with Gromacs 4.6.5 51 and the Amber99sb forcefield, 47 which was shown to perform well in the prediction of NMR results. 52 Particle mesh Ewald was used to include long-range interactions with a non-bonded cut-off set to 13.5 Å. Hydrogen bonds were constrained using the SHAKE algorithm. The time step was 2 fs and trajectory files were written out every 20 ps. The temperature was controlled using the velocity-rescale algorithm. In the bright state, His193 was protonated and the chromophore was in the anionic form, while in the dark state the chromophore was turned into the neutral form and His193 was neutral as well (with the proton on Nδ). 28 For the simulation, all of the crystallographic waters were maintained, since they play an important role in the chromophore surrounding. The charged protein was neutralized by Na+ and Cl- ions at a concentration of 0.15M and solved in a cubic TIP3P water box, which was 10 Å bigger (in each direction) than the dimensions of the protein. Each chromophore transformation and the mutations were made on the original crystal structure. Next, the system was minimized (steepest descent method followed by L-FBGS) and equilibrated with all the heavy atoms being restrained, at T=300K and 1bar (100ps). It was then passed to the production run with no constraints for 100 ns. For each protein, 5 independent equilibration and production runs were performed, resulting in 5×100 ns of statistically independent simulations.

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Flexibility estimation RMSF calculation For the estimation of protein flexibility, the RMSF of the backbone was measured. The first 20 ns were cut out of each trajectory, and the trajectories were aligned on the first frame. The average RMSF was calculated over 400 ns. PLS-based FMA calculations To probe which residues have an influence on the photoswitching properties, we performed a Partial Least-Squares based Functional Mode Analysis (PLS-FMA). 53 These calculations detect the principal collective mode which correlates with a given functional property of the protein, in our case photoswitching. Since the RSFPs assume either a bright or a dark state, we label these states as +1 and -1, respectively. PLS-FMA works by taking the trajectory of the simulation and using half of it as a training set and half as a model set. Hence we mixed the trajectory for the bright and dark protein making sure the training set contains both trajectories (i.e. first 200 ns from the bright protein trajectory, then 200 ns from the dark protein, then again bright and dark). An example of the correlation between the training and cross-validation model set can be seen in Fig. S3. This calculation resulted in ensembleweighted maximum correlated motions (ewMCM) trajectories for different pairs of proteins, which are discussed in the Results section. To avoid the problem of overfitting, we trained the model to up to 50 components (see Fig S2). Using the Pearson correlation coefficient we concluded that the best fit occurs for 5 components with a correlation coefficient of almost 94%, after which the correlation on the test set decreases due to overfitting.

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Results and Discussion RMSF as a criterion for the photoswitching property It has been reported that the flexibility of the FP β-barrel is dramatically affected by the photoswitching. 39 This finding, based upon NMR studies, states that the dark-state Dronpa has higher flexibility around the hydroxyphenyl ring of the chromophore and the neighboring β-strand. In particular, the aminoacids in front of the chromophore’s hydroxyphenyl ring, namely in β-sheets 7, 8, 9, 10 and 11 (see Fig. 1C), show broadening of the NMR signal due to a higher flexibility of this region, occurring on a timescale that is short compared to the NMR relaxation time. In the bright state, the chromophore is tethered to the β-barrel via a hydrogen bond with Ser142 and a stacking interaction with protonated His193. In the dark state, however, this connection is lost, allowing those and neighbouring aminoacids to gain more flexibility. Also, the hydrogen bonding between β strands 7 and 10 is weak, further adding to the dark state flexibility. Broadening of the spectral signatures of aminoacids 65-69, in the α-helix holding the chromophore and adjacent to the loop 3, was also observed. The results of the MD simulations and the RMSF flexibility analysis are represented in Fig. 2 for Dronpa and Fig. S4 and S5 for IrisFP and pcDronpa, respectively. From these data, it is clear that for all three proteins the RMSF of the dark state on average is indeed higher than that of the bright state. Taking a closer look, we can see that the increased flexibility of the dark state is situated in the β-sheets close to the chromophore’s hydroxyphenyl moiety, especially around the β-strands 7 and 10 and the α-helix (see Fig. 1C for the arrangement of the β-sheets). Furthermore, the Dronpa trajectories show that the higher flexibility is mostly due to the rearrangement of the His193, Arg66 and also sterical hindrance of the chromophore by the protein environment around residues Val157, Phe173 and Glu140. A noteworthy feature is the higher flexibility that is observed at position Tyr116 and Asn105. If we look into the structural rearrangement between the bright and dark state we arrive 9



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to the conclusions observed earlier: 40,54 particularly strong H-bond between Ser142 and the chromophore in Dronpa bright and absence of such a bond in Dronpa dark. Also, Dronpa bright has a stable H-bond network formed around the chromophore through residues Glu212 – His193 – Glu144 – Arg66 (Fig. S6). In Dronpa dark this network is lost and the chromophore possesses higher flexibility, due to the isomerization of the chromophore and deprotonation of His193 28 (Fig. S7). Also due to the transient repulsion during the isomerization of the chromophore, Val 157 flips by 100 thus giving way to accommodate deprotonated ◦

His193. The latter serves as a H-bond acceptor for Ser142, although this bond is not very stable since Ser142 forms a second H-bond with the backbone atom of Glu140 as well, thus giving rise to flexibility in the β-sheets 7 and 10. Next, we compared EosFP, considered non-switchable, to IrisFP. In Fig S3 it can be seen that the average flexibility of the dark-state of IrisFP is only slightly higher compared to the bright state. In β-sheets 4, 8, and 9, adjacent to the Ser173 which introduced the switching compared to EosFP, the flexibility of the dark-state is comparable to the flexibility of the bright-state. This is in agreement with the explanation for the effect of the Phe173Ser mutation on the photoswitching properties, 15 namely the replacement of Phe173 by Ser, resulting in a conformational change of the Met159 residue and the creation of a larger cavity. Also less tight packing of the cavity around the chromophore allows higher flexibility for Ser173. That leads to higher flexibility of the chromophore which is in line with the observed lowering of the QY 55 (also see the comparison of the chromophore’s flexibilities in Fig. S8A). Correlation between the Phe173 conformation and the chromophore state in Dronpa was also observed by Moors et al. 29 This observation about the flexibility in the β-sheet 7 and 10 for Dronpa also holds for IrisFP, which has structural rearrangement very similar to Dronpa. We also studied pcDronpa and compared its dark state flexibility to Dronpa and IrisFP. The dark-state of pcDronpa has an even higher flexibility than it does in Dronpa and IrisFP (see Fig. S4). This can be explained by the combination of features of both the IrisFP and

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Dronpa environment. pcDronpa has a chromophore which is identical to IrisFP’s and the sequence and structure are very similar to Dronpa’s. From this, we can conclude that in Dronpa-like photoswitching proteins (where excitation light also brings the FP to the dark state) 56 the dark-state indeed possesses higher flexibility in β7-10 sheets. This most probably results from a lower stabilization of the trans-state by the protein environment (which also possibly contributes to a higher energy in comparison to the cis-state). This observation is in line with the conclusions made about IrisFP and EosFP. 15,55 In this study authors assign higher enthalpy to the dark state with the difference of 68 kJ/mol for IrisFP and 114 kJ/mol for EosFP. Another study on asFP595 (a protein which contrary to Dronpa is formed in the dark state and excitation light brings it to the bright state) by Mironov et al. presents the free energy difference upon thermal isomerization of the chromophore. The stable dark-state has a free energy difference of ≈ 25 kJ/mol in comparison to the bright-state. 38 IrisFP has a higher flexibility than Dronpa, but also a lower molecular brightness (27.9 mM-1 cm-1 and 72.2 mM-1 cm-1 respectively). 15,40 The same applies to pcDronpa which has a flexibility in the bright state similar to that of Dronpa as well as a similarly high molecular brightness (97.8 mM-1 cm-1 ). 17 We notice increased RMSF in rsKame in the bright-state and accordingly it also has lower brightness than Dronpa. 41 Hence there indeed seems to be a correlation between brightness and flexibility in the bright state (see Fig. S8B). Ultimately high flexibility could be one of the reasons of fluorescence absence in the dark-state.

Mapping collective protein motions associated with the photoswitching Although our RMSF calculations clearly show that the flexibility of the RSFP’s β-barrel depends on their state, these techniques lack the sensitivity to distinguish which aminoacids are crucial for triggering these differences. One way to identify the aminoacids contributing the most to the inflated flexibility in 12


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most promising candidates to either improve the photoswitching property or to inhibit it. Our PLS analysis clearly distinguishes two separate regions of interest: the first one consists of β-sheets 7,8, 9 and 10 which are situated adjacent to the chromophore’s hydroxyphenyl ring, the second region being the α-helix holding the chromophore. Particularly in IrisFP, Ser173, responsible for the photoswitching properties, has a high ewMCM, just like Ile157 and to lesser extent His193. The high ewMCM around Gly155 is most likely the result of glycine’s lack of side chain. In Dronpa, the higher ewMCM values are found at residues Val157, Glu144 and Tyr116 while in pcDronpa Ser142, Met159 and His193 show high ewMCM values. These residues are known to be involved into the process of photoswitching and as can be seen from Fig. 3, are mostly located in β8. 56 Also, it is known that mutations in the α-helix influence the photophysical properties of RSFPs. In particular the A69T mutation in pcDronpa significantly lowers its brightness and alters its pKa, 17 while the V60A mutation in Dronpa produces a "fast-forming" variant with an increased brightness. 17 In our case aminoacids 65-68 also contribute to the difference in promoting motions of the protein in bright and dark state. The results obtained by the PLS-based FMA confirm that there is a significant difference between pcDronpa, Dronpa and IrisFP in loop 3 (see the loop numbering in the Fig.

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which seems to contribute the most to the photoswitching property change in Dronpa and pcDronpa, while in IrisFP this movement seems to be distributed more over the two other loops. This can be explained by the fact that in loop 2 in IrisFP, there is one extra Gly residue (Gly180) that is not present in in Dronpa and pcDronpa. This makes loop 2 longer and more flexible. Also, at position 73 (which is in loop 3, following the Dronpa numbering) IrisFP has Gln, while in Dronpa and pcDronpa it is Val, which does not form a stable Hbond with Asp184 (corresponding to Lys183 in case of IrisFP). It is interesting to note how conformational changes in the loops are connected with the chromophore’s conformational change. Loop 3 in particular is connected with the α-helix and residues 65-69 adjacent to the chromophore, so its higher flexibility in the trans-state may contribute to the higher

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displacement of the chromophore. The same aminoacids in the α-helix show broadening of the NMR signal in the dark state Dronpa. 39 Another aminoacid of interest in Dronpa is Tyr116, which is Asn in IrisFP and pcDronpa. Remarkably, this aminoacid was mutated from Tyr in pcDronpa, yielding pcDronpa2 which has an improved green-to-red photoconversion rate. The interaction of the aminoacid at position 116 with the chromophore is indeed different between Dronpa and pcDronpa/IrisFP. In case of Dronpa the CYG chromophore forms H-bonds with Tyr116 through a water molecule, while in pcDronpa and IrisFP the HYG chromophore interacts with Asn116 sidechain through two coordinated water molecules. The PLS-FMA clearly identifies several of the known aminoacids participating in the modulation of the photoswitching properties. Also it has added a new dynamic level to the study of the structural properties of the RSFPs. We elaborated on this concept by testing whether our technique can probe the effect of point mutations on the flexibility of RSFPs. The results are presented in the following section.

Single-point mutation effect on the flexibility of RSFP Although the previous section has proved that PLS-FMA can contribute to identifying the aminoacids which are determining the photoswitching properties we would like to fine-tune the interpretation of the results which we obtained from it. The easiest way to do so is to test the RMSF/FMA protocol on a single-point mutant set. Also by doing so we have systematized the difference in flexibility which was noted in the previous section and connected it to the photo-physical properties. From the previous experiments, we found that in the Dronpa-like switching RSFPs the dark state has a higher flexibility while the FMA analysis pointed out the key aminoacids which promote the ewMCM in dark or bright state, i.e. promote the difference in the motions in the two states. We now investigate how the flexibility of Dronpa changes in its fastswitching mutant rsFastLime and a slow-switching mutant rsKame. The basic explanation 15



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of the difference in the QY of the photo-isomerization seems straightforward: Gly157 is a small aminoacid hence it makes a bigger cavity around the chromophore and in such a way decreases the steric hindrance for a cis-trans isomerization of the chromophore. 12 In the case of rsKame, Leu157 is a slightly more bulky aminoacid in comparison to Val, hence it increases the sterical hindrance when the chromophore undergoes the isomerization. 41 However, comparison of the effect of the single V157G/L mutation on the flexibility in Dronpa, rsFastLime and rsKame in their bright and dark state does not indicate which of those 3 proteins would be a faster photo-switcher. Both "fast" and "slow" switchers have increased flexibility in the dark state in comparison to Dronpa (see results in Fig. S7), and rsKame shows higher flexibility in the bright-state as well. Apparently the switching mechanism itself may play a role and we therefore investigate if the speed of dark-to-bright switching is hindered by sterical arrangement of the aminoacids around the chromophore. We have checked whether the rate of isomerization may be dependent on the intermediate state when the protein environment is still not fully adapted to the chromophore. Recent experimental studies on the photoswitching mechanism of Dronpa by van Thor et al. as well as Yadav et al. 30,58 indicate that the first step for the photoswitching process is the isomerization of the chromophore which is followed by ground-state proton transfer. Here, following the notion of Andresen et al. about the sterical hindrance of the chromophore by Val157, 40 we have tried to mimic this situation which we will denote as a transient state. In the transient state, the chromophore is already in its neutral trans-state, while the protein is not yet fully adjusted and still corresponds to the bright-state. Also the His193 side chain is in its neutral form (proton on Nδ). Then we run the MD simulation where during the first 10-20 ns (see Fig. S9) the most important aminoacids like Arg66 and His193 change their conformation to the one which is closer to that of a dark-state protein. So although all of the events related to the photoswitching have already happened the protein is still not fully relaxed (here we are talking about the relaxation times taking more than microseconds) in relation to the crystal dark state.

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Mimicking a transient dark state will allow us to compare these results to the nonswitching proteins where the dark-state simply doesn’t exist so that we cannot make use of the available crystal structure (i.e. in EosFP). First, we checked whether the simulated transient structure adjusts to the dark crystal structure and we found that RMSD from the Dronpa dark crystal structure is ≈0.5 nm2 , while for original dark state the RMSD is ≈0.2 nm2 . We noticed that the concerted motion of His193 and Arg66 takes place during the first 10-15 ns of the simulation for Dronpa and rsFastLime, while for rsKame it takes more than 20 ns (see Fig S8). The flexibility of the backbone in the simulated transient states is represented in Fig. 4A. The results show us that in the transient dark-state, the mutated proteins’ (rsKame and rsFastLime) flexibility does differ in comparison to Dronpa. Even more, we observe a higher flexibility for rsKame (slow-switcher) in the transient dark state, while for rsFastLime the difference is almost not present, since the protein quickly converges to the natural dark-state structure. The resulting difference between the flexibility in the bright and transient dark state can be found in the Fig. 5. It is plotted against the on-off photo-isomerization quantum yield (QY), although this observation does not necessarily imply a direct causal relationship. Indeed rsFastLime has the highest QY but the smallest difference in the flexibility, while for rsKame the opposite holds place. Also if we look at the off-state relaxation half-times then for Dronpa it is 840 min and for rsFastLime – 8 min. 12 Although not being reported explicitly, Rosenbloom et al show that rsKame has a longer dwell time in the dark state. 41 All of this points to a higher energy difference between the dark and bright state in the slow-switching RSFP. The results of the FMA analysis on rsKame and rsFastLime can be found in Fig. 6. In the case of rsFastLime, the collective motions of the bright and dark states have very few differences, while in case of rsKame the amplitude of ewMCM is much higher, meaning that the bright and dark states have very different functional modes. The differences between collective motions arise from the difference in the energy landscape of the two states. The

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Figure 5: The quantum yield of the on-off isomerization plotted vs. the difference in the flexibility between the transient dark and the bright state. The quantum yields are taken from literature and the flexibility difference is calculated for the chromophore and the aminoacids situated in the β-sheets in front of it, namely 135-144, 150-159, 166-174, 186-194 and 206-212 following Dronpa numbering. transition state and fall into the second state. Hence we can say that if two states are close, the photoswitching will occur faster. This leads us to conclusion that there is a link between the difference in the collective motions and the speed of the photoswitching. However their connection would be rather phenomenological than direct. Combining the FMA and RMSF results in both the natural and transient dark states we come to two conclusions. First of all, in rsFastLime both the bright and dark state are very close energetically and are separated only by a low energy barrier. Secondly, for rsKame the bright state is energetically less favourable than in Dronpa, while its dark state is separated by a high energy barrier. Most probably in case of rsKame there is more than one transition state each corresponding to a change in a principal mode differentiating the bright and dark states. Another pair of close mutants we have looked at is IrisFP and EosFP. Although IrisFP is a mutant of EosFP, we have also tried to compare EosFP to the results of back-mutation S173F introduced into IrisFP. The combinations used for the analysis can be found in Fig. 19




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similar to that of EosFP. Hence we calculated the flexibility of the transient state of IrisFP S173F. Its flexibility is higher than in the case of dark state IrisFP S173F and shows a behavior more similar to EosFP. If we look at the flexibility of EosFP in the bright and dark state then we can say that the latter one is substantially higher in the β-sheet 7, 8, 9 and 10, located around Phe173, Ile157 and Glu140, His193. All these residues are able to sterically hinder the chromophore’s isomerization. Also, the average flexibility of EosFP in the dark state is higher than that for any other RSFP. The difference between the bright and transient dark state for IrisFP and EosFP is shown on the Fig. 5. Like in the case with Dronpa it can be noticed that the difference is much higher for non-switching EosFP than for IrisFP. Combining the results of RMSF and FMA for single-point mutants of EosFP and Dronpa it becomes apparent that the effect of a single mutation can barely be seen when comparing it only in the existing crystal structures. PDB structures provide static picture where the protein environment can easily incorporate the mutation, but photo-isomerization is a highly dynamic process involving many intermediate states on the way from the bright to dark state. That is why transient states can play a role when trying to predict the effect of the mutation. Although they correspond only to the ground state, there might be a phenomenological link between transient-bright state difference and the QY of the photo-isomerization. In fact thermal relaxation is a ground-state process and the transient state can be contributing to the reduced photo-isomerization QY by constantly repopulating the bright state.

Conclusions In this paper we have investigated flexibility and functional mode sensitivity as dynamic indicators of photoswitching properties in Dronpa, IrisFP and several of their mutants. We found that for Dronpa and its photoconvertible mutant pcDronpa, the flexibility, as quantified by RMSF analysis, is considerably higher in the dark-state, while for IrisFP this effect

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is limited to only two β-sheets close to the chromophore. Furthermore, we can see that the flexibility of EosFP in the dark-state is high as well, in fact much higher than in Dronpa, IrisFP or pcDronpa. This can be explained by the fact that normally the dark-state in Dronpa-like RSFPs has a higher energy in comparison with the bright-state. As a result, in non-photoswitchable FPs the dark state is simply not or barely populated due to a too high energy difference separating two states. Increased flexibility can relate to the lower stabilization of the H-bond network between the aminoacids, so although the dark-state is virtually possible in such protein as EosFP, it is not long-lived and the chromophore quickly relaxes to the bright-state structure. This hypothesis is supported by the fact that the final dark states of the RSFPs have lower flexibility than the transient states connecting the bright and dark states. We observed a correlation between the brightness of fluorescence and the flexibility of the bright-state, not only for Dronpa, pcDronpa and IrisFP, but also for the Dronpa mutants rsFastLime and rsKame. The FMA proves to be a sensitive tool to probe mutation sites, which would affect the most the photoswitching property of the protein. Specifically for Dronpa the sites of interest are the aminoacids situated in the β-sheets 7-10 and also the ones in the loop connected to the α-helix.

PLS-based FMA analysis of the effect of a single mutation

(Dronpa/rsFastLime/rsKame) shows a striking difference in the results. In particular in rsFastLime the difference in the collective motions between bright and dark state is barely noticeable and involves mostly the α-helix. In rsKame it is much higher and is brought about by the β-sheets 7-10. Dronpa is situated somewhere in between these two and shows high difference in the motions of Val157. Thus the lower difference in the internal collective motions of the bright and darks state of RSFP could contribute to the faster-switching property. In conclusion, flexibility analysis of RSFPs correlates with photoswitching properties, but as an indicator provides only a low resolution measure for protein design. On the other

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hand FMA yields complementary information to predict mutation sites that have a high probability to modulate photoswitching properties. Further studies involving the study of the free energy difference and the detailed study of the mechanism involved into the process of photoswitching both thermally and photochemically are needed to understand the whole picture.

Acknowledgement Financial support from the Flemish Government through the KU Leuven concerted action scheme is gratefully acknowledged. The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Hercules Foundation and the Flemish Government. B.M. is funded by a Ph.D. grant from the Agency for Innovation by Science and Technology (IWT) Flanders. P.D. thanks the Research Foundation-Flanders (FWO Vlaanderen) for a postdoctoral fellowship and the KULeuven for a research professorship.

Supporting Information Available Chromophore’s parameters and flexibility anlysis for all the proteins which were discussed above. This material is available free of charge via the Internet at http://pubs.acs.org/.

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Molecular Dynamic Indicators of the Photoswitching Properties of

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