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Jan 12, 2018 - (ArcLight) whose mechanism is currently under debate. Fluorescent proteins1,2 (FPs) are commonly used to probe biological processes by ...
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Fluorescent proteins detect host structural rearrangements via electrostatic mechanism Lena Simine, Heiko Lammert, Li Sun, Jose Nelson Onuchic, and Peter J Rossky J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10851 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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Fluorescent proteins detect host structural rearrangements via electrostatic mechanism Lena Simine1*, Heiko Lammert3, Li Sun3, José N. Onuchic1,2,3, Peter J. Rossky1* Departments of Chemistry1, and Physics2, and the Center for Theoretical Biological Physics3, Rice University, Houston, Texas 77030

E-mail: [email protected], [email protected] Supporting Information Placeholder The rational design of genetically encoded fluorescent biosensors, which can detect rearrangements of target proteins via interdomain allostery, is hindered by the absence of mechanistic understanding of the underlying photo-physics. Here, we focus on the modulation of fluorescence by mechanical perturbation in a popular biological probe: enhanced Green Fluorescent Protein (eGFP). Using a combination of molecular dynamics (MD) simulations and quantum chemistry, and a set of physically motivated assumptions, we construct a map of fluorescence quantum yield as a function of a 2D electric field imposed by the protein environment on the fluorophore. This map is transferable between Tsien’s Class 2 GFP’s, and it allows one to estimate the shifts in fluorescence intensity due to mechanical perturbations directly from MD simulations. We use it in combination with steered MD simulations to put forward a hypothesis for the mechanism of a genetically encoded voltage probe (ArcLight) whose mechanism is currently under debate. Fluorescent proteins1,2 (FPs) are commonly used to probe biological processes by fusing them with proteins of interest and expressing them in vivo. For the class of biological probes that we are concerned with in this paper, the target protein undergoes a structural rearrangement in response to a stimulus, for example, binding of ATP/ADP3, binding of Ca2+ ions4. This rearrangement modulates either the brightness or hue of FP’s fluorescence via interdomain allostery. Circularly permuted Green Fluorescent Proteins (cpGFPs) are commonly used as reporters3–6, because of the proximity of the fusion sites (termini) to the chromophore. This proximity ensures that the rearrangement of the fusion protein affects the chromophore directly. Interestingly, effective probes based on the topologically conventional variants of GFP have also been constructed. For example, the FP in ArcLight voltage probe7 reports the rearrangement of its host with a strongly dimmed fluorescence signal. A consistent mechanism of fluorescence quenching in this class of sensors was conjectured to be related to a change in the protonation state of the chromophore4,8, but neither a conclusive evidence, nor a more detailed mechanism for it have been established. Understanding the mechanism would allow one to devise strategies to improve existing probes, and further guide the development of FP-based biosensors. We set a dual goal for this Communication: 1. To develop a computationally efficient method to (crudely) relate FP’s conformation to fluorescence intensity for rapid and cost-effective screening of potential probes, and 2. To suggest electrostatic control of fluorescence as a relevant mechanism for sensing. As a case-study, we investigate the possibility that this mechanism accounts for the poorly understood behavior of ArcLight.

Figure 1. An illustration9 of the suggested mechanism of fluorescence quenching in the ArcLight probe: the fluorescent protein (green, gray) is quenched as a result of a mechanical compression against the membrane by the structural rearrangement of its host, the voltage sensing domain (orange), which happens in response to membrane (purple) depolarization. ArcLight, a genetically encoded voltage probe, is used to detect action potentials in neurons7. It is composed of voltage sensing domain (VSD), a membrane protein which undergoes a structural rearrangement in response to the action potential, and a FP whose fluorescence is then quenched. We propose that the fluorescence in ArcLight is dimmed because the FP is mechanically deformed by the rearrangement of its host protein, the VSD10. In order to devise a specific reaction coordinate for this process, we integrated the available experimental data8,10, and conclude that FP’s compression against the membrane, in which the termini are brought closer together is a plausible outcome of VSD’s rearrangement. Fig. 1 shows a schematic representation of this process. For a more detailed discussion of the experimental data, see Supporting Information. Since it has been shown that the excited state relaxation in ArcLight is largely unaffected by the external fields11, we focus on the effects of local rearrangements of the residues in the vicinity of the chromophore within the protein as a result of this compression. We use the enhanced (e)GFP as our model system.

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Fluorescent proteins evidently suppress the spontaneous nonradiative relaxation of excited chromophore. In order to understand how this happens a significant progress has been made recently in identifying the appropriate computational protocols for modelling of excited state dynamics of chromophores in fluorescent proteins13–18. Here, we leverage these findings in order to

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construct a model that can capture the modulation of fluorescence intensity by mild mechanical perturbations of the protein. Our expectation is that a mechanical perturbation of the protein results in a rearrangement of the residues which come in contact with the chromophore and affects the excited state relaxation behavior. Similar scenarios have been previously discussed in the context a mechanical perturbation of FP’s by hydrostatic pressure19,20 with encouraging, yet not readily generalizable results. Since one of our goals is to generate a practical screening tool, we will construct a computational model21 for an entire class of fluorescent proteins used for voltage sensing (e.g., ArcLight), namely, Class 2 in Tsien’s classification1. In proteins which belong to Class 2, our model system eGFP, the chromophore exists predominantly in a deprotonated form. We, therefore, limit our analysis to the anionic–hydroxybenzylidene-2,3, dimethylimidazolinone, see Fig 2(b). For clarity, we will refer to it simply as “the chromophore” hereafter.

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Figure 2. (a) A schematic of the proposed uni-axial compression reaction coordinate. The arrows point at the “anchor” residues (K3 and A227, PDB 4EUL12). D indicates the equilibrium distance between them in the native (left) and compressed (right) conformations of eGFP, (b) A typical Franck-Condon (top), and a transition state (TS) (bottom) geometries of the anionic GFP chromophore along the HulaTwist trajectory. The atoms are color-coded: N(blue), O(red), C(gray), H(light gray). The axes X (red) and Y (blue) point in the positive directions of the electrostatic potential differences, ∆𝐸 x and ∆𝐸 y. The letters (OP, OI, C and N) label the atoms used in the calculation of ∆𝐸 x and ∆𝐸 y. (c) Free energy calculated as the potential of mean force (PMF) for ∆𝐸 x and ∆𝐸 y. The mean distances D between the anchor residues are indicated on the figures for the native (top panel), and the uni-axially compressed eGFP (bottom panel). Note that the panels share the x-axis; the dashed lines pass through the minima of the free energies to emphasize relative shifts.

Fig. 2(a) shows the two conformations of eGFP that we will compare: native, and uni-axially compressed. The conformations are characterized by the distance (D) between the “anchor” residues (K3 and A227, PDB 4EUL12). The former represents an OFF state of the ArcLight complex: the native and bright conformation of the eGFP. The latter represents ArcLight’s ON state, in which the eGFP is uni-axially compressed against the membrane by the VSD. In order to be consistent with experimental observations, the fluorescence in the ON conformation needs to be dimmer than in the OFF state. Next, we develop the theoretical framework necessary to check this consistency.

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Figure 3. (a) Estimated free energy of non-radiative decay along the hula-twist trajectory from the Franck-Condon geometry to the transition state (see Fig. 2b), based on a set of protein conformations sampled from simulations of the eGFP in the native basin (green), and of uni-axially compressed eGFP (black). The S1 potential energy surface for an isolated chromophore in vacuum is shown in brown. (b) Quantum yield as a function of ∆𝐸 x and ∆𝐸 y. Red circles mark the two hypothetical eGFP conformations representing the ON and OFF states of the ArcLight sensor: native and uni-axially compressed. The importance of electric fields in fluorescent proteins was emphasized in earlier theoretical22 and experimental work23 and some of the effects of the polarizing environment on the excited state behavior of the chromophore have been uncovered in recent atomistic simulations. For example, Morozov and Groenhof15 point at hydrogen bonding to the phenolate oxygen of the chromophore as a source of polarization sufficient to keep it from relaxing via a non-radiative decay. Park and Rhee14 demonstrate that electric fields modify the shape of the excited states potential surfaces. They conclude that the non-radiative relaxation is controlled by these fields and not by steric hindrance due to surrounding residues. These results form the starting point for our work. In the interests of further generalization of these findings we consider here whether electrostatic environment, irrespective of chemical, may be meaningfully captured with only a few coordinates. Representing the environment through coordinates based on electrostatic potential (ESP) allows us to depart from sequencespecific representations, and draw conclusions which may apply to classes of sequences. First, we note that the excited state relaxation process involves a concomitant migration of charge density away from chromophore’s phenolate ring14,15, which is likely affected by the external electrostatic environment. Being a planar molecule the chromophore is polarizable along two dimensions, see Fig. 2(b): the long axis X, and the short axis Y. We define the relevant coordinates by the differences of ESP along these polarization axes. Practically, we compute the potential due to the surrounding partial charges assigned by the force-field at the locations of atoms which are indicated on Fig. 2(b) with letters. We then take the difference between pairs of potentials at locations oriented along X and Y, to obtain the approximate linear gradients: ∆Ex and ∆Ey. For discussion see Supplementary Information. Next, we quantify the effect of polarization on the outcome of the excited state relaxation of the chromophore by computing the quantum yield. To this end, we approximate the excited state (S1) free energies along a non-radiative decay reaction coordinate. The relaxation path of the excited chromophore in a protein is a controversial topic14–16,24, and we chose a reaction coordinate with the intention to avoid this controversy. Specifically, we have adopted the initial fragment of a volume conserving hula-twist15 leading up

to the energy barrier separating the bright and the dark basins (see Fig. S4 in Supplementary Information). This choice restricts our theory to Class 2 GFP’s undergoing excited state relaxation through a set of paths which start with the rotation of the phenolate ring including paths in which only the phenolate ring is rotating as well as the hula-twists. To the best of our knowledge, the only other alternative path (the relaxation through the rotation of the imidazole) is suppressed inside protein, and our reaction coordinate captures the physics necessary for the construction of a qualitative theory. Fig 3(a) shows that the protein environment lifts the barrier by several kBT in the native eGFP (shown in green), which is consistent with the fact that eGFP is fluorescent and bright. Since the barriers are very high, we resort to Arrhenius theory to approximate the rates of non-radiative decay, 𝑘$% = 𝐴𝑒 )*+ /-. / , where Eb is the transition state free energy gap, kb is the Boltzmann constant, T is temperature, and A is the rate pre-factor which captures the time-scale of the thermalization process. We obtain phenomenologically the pre-factor A, and the radiative rate (𝑘% ) from eGFP’s known fluorescence life-time and quantum yield. Finally, we assume that A and 𝑘% do not change significantly within the range of electric fields considered here, and estimate the quantum -6 yield, 𝑄𝑌 = , on a 5x5 grid of ∆𝐸9 and ∆𝐸: , Fig. 3(b). A -6 7 -86

simple moving-window interpolation was used on the crude data. For full details of this calculation, as well as for a discussion of the invoked approximations, see Supporting Information. We are now in a position to check whether our hypothesis for the mechanism of ArcLight is consistent with experimental observations7. The expectation is that fluorescence will be quenched in the uni-axially compressed conformation, which corresponds in our theory to the ON state of the sensor. First, we compare the free energies characteristic of native, and compressed eGFP. To this end, we construct our potentials of mean force (PMF’s) using the probability distributions of ∆𝐸 x and ∆𝐸 y, obtained using molecular dynamics simulations (see Supporting Information): 𝑃𝑀𝐹 = −𝑘? 𝑇 log 𝑃 ∆𝐸9 , ∆𝐸: . The two panels in Fig. 2(c) show the free energy of native eGFP (top panel), and that of the compressed eGFP (bottom panel). The shifts of the free energy

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minima due to a mild mechanical compression are quite significant along both axes (~1V along X, and ~0.5V along Y): the native basin of eGFP imposes a relatively strong potential energy drop across the long axis of the chromophore (X) stabilizing the negative charge on the phenolate oxygen, while the polarization along the short axis (Y) is small. In contrast, the electric field in the compressed eGFP exhibits a vanishing component along X, and a negative gradient along Y.

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We obtain the approximate free energies along the non-radiative decay reaction coordinate in these two conformations. Fig. 3(a) shows that the height of the energy barrier is strongly affected by the shift of the polarizing potential. In the native eGFP, the electric field lifts the barrier by ~10kBT, relative to vacuum (brown). In contrast, in the uni-axially compressed eGFP, a much smaller barrier appears, ~3kBT. This difference in the free energy barrier heights corresponds to two orders of magnitude faster nonradiative decay in the compressed conformation compared with native. We demonstrate the corresponding shift in fluorescence brightness by locating the relevant points on the quantum yield map: see the red circles on Fig. 3(b). These results show that our hypothesis for the mechanism of ArcLight is consistent with experimental observations.

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Supporting Information Theoretical methods and procedures.

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AUTHOR INFORMATION

https://creativecommons.org/licenses/by-nc-sa/4.0/. Li, Q.; Wanderling, S.; Paduch, M.; Medovoy, D.; Singharoy, A.; Mcgreevy, R.; Villalba-galea, C. A.; Hulse, R. E.; Roux, B.; Schulten, K.; Kossiakoff, A.; Perozo, E. Nat. Struct. Mol. Biol. 2014, 21, 244–252. Brinks, D.; Klein, A. J.; Cohen, A. E. Biophysj 2015, 109, 914– 921. Arpino, J. A. J.; Rizkallah, P. J.; Jones, D. D. PLoS One 2012, 7, e47132–e47132. Maddalo, S. L.; Zimmer, M. Photochem. Photobiol. 2006, 82, 367–372. Park, J. W.; Rhee, Y. M. J. Am. Chem. Soc. 2016, 138, 13619−13629. Morozov, D.; Groenhof, G. Angew. Chemie Int. Ed. 2016, 55, 576. Snyder, J. W.; Parrish, R. M.; Martínez, T. J. J. Phys. Chem. Lett. 2017, 8, 2432. Olsen, S.; Mckenzie, R. H. J. Chem. Phys. 2009, 130, 184302. Sinicropi, A.; Andruniow, T.; Ferre, N.; Basosi, R.; Olivucci, M. J. Am. Chem. Soc. 2005, 127, 11534–11535. Mauring, K.; Deich, J.; Rosell, F. I.; Mcananey, T. B.; Moerner, W. E.; Boxer, S. G. J. Phys. Chem. B 2005, 109, 12976–12981. Laurent, A. D.; Mironov, V. A.; Chapagain, P. P.; Nemukhin, A. V; Krylov, A. I. J. Phys. Chem. B 2012, 116, 12426–12440. We emphasize that our theory is not intended for quantitative prediction of quantum yields or for ranking of different sequences by brightness. We seek to compare the changes in quantum yields of different conformations of the same protein. Martínez, T. J. Faraday Discuss. 2004, 127, 227– 266. Mandal, D.; Tahara, T.; Meech, S. R. J. Phys. Chem. B 2004, 108, 1102–1108. Martin, M. E.; Negri, F.; Olivucci, M. J. Am. Chem. Soc. 2004, 126, 5452–5464.

Corresponding Author E-mail:

[email protected],

[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was enabled by NSF grant CHE-1641076, and the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1053575 (35). Additional support was provided by the Center for Theoretical Biological Physics sponsored by the NSF (Grant PHY- 1427654) and by NSF- CHE 1614101. LS thanks Igor Polyakov and Loic Jouber-Doriol for initiating LS into CASSCF. We thank Gerrit Groenhof for providing the hulatwist trajectory, and Anna Krylov for a critical reading of the manuscript.

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Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 544. Acharya, A.; Bogdanov, A. M.; Grigorenko, B. L.; Bravaya, K. B.; Nemukhin, A. V; Lukyanov, K. A.; Krylov, A. I. Chem. Rev. 2017, 117, 758−795. Berg, J.; Hung, Y. P.; Yellen, G. Nat. Methods 2009, 6, 161– 166. Nagai, T.; Sawano, A.; Park, E. S.; Miyawaki, A. Proc. Natl. Acad. Sci. 2001, 98, 3197–3202. Belousov, V. V; Fradkov, A. F.; Lukyanov, K. A.; Staroverov, D. B.; Shakhbazov, K. S.; Terskikh, A. V; Lukyanov, S. Nat. Methods 2006, 3, 281–286. St-pierre, F.; Marshall, J. D.; Yang, Y.; Gong, Y.; Schnitzer, M. J.; Lin, M. Z. Nat. Neurosci. 2014, 17, 884–889. Jin, L.; Han, Z.; Platisa, J.; Wooltorton, J. R. A.; Cohen, L. B.; Pieribone, V. A. Neuron 2012, 75, 779–785. Han, Z.; Jin, L.; Chen, F.; Loturco, J. J.; Cohen, L. B.; Bondar, A.; Lazar, J.; Pieribone, V. A. PLoS One 2014, 1–21. www.somersault1824.com. Creative Commons license for graphics (CC BY-NC-SA 4.0)

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