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Proton Wire Dynamics in the Green Fluorescent Protein Ai Shinobu, and Noam Agmon J. Chem. Theory Comput., Just Accepted Manuscript • DOI: 10.1021/acs.jctc.6b00939 • Publication Date (Web): 15 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

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Journal of Chemical Theory and Computation

Proton Wire Dynamics in the Green Fluorescent Protein Ai Shinobu and Noam Agmon* The Fritz Haber Research Center, Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel email: [email protected]

Abstract Inside proteins, protons move on proton wires (PWs). Starting from the highest resolution X-ray structure available, we conduct a > 300 ns molecular dynamics simulation of the (A state) wild-type (wt) green fluorescent protein (GFP) to study how its PWs change with time. We find that the PW from the chromophore via Ser205 to Glu222, observed in all X-ray structures, undergoes rapid water molecule insertion between Ser205 and Glu222. Some times, an alternate Ser205-bypassing PW exists. Sidechain rotations of Thr203 and Ser205 play an important role in shaping the PW network in the chromophore region. Thr203, with its bulkier sidechain exhibits slower transitions between its three rotameric states. Ser205 experiences more frequent rotations, slowing down when the Thr203 methyl group is close by. The combined states of both residues affect the PW probabilities. A random walk search for PWs from the chromophore reveals several exit points to the bulk, one being a direct water wire (WW) from the chromophore to the bulk. A longer WW connects the “bottom” of the GFP barrel with a “water pool” (WP1) situated below Glu222. These two WWs were not observed in X-ray structures of wt-GFP, but their analogues have been reported in related fluorescent proteins. Surprisingly, the high-resolution X-ray structure utilized herein shows that Glu222 is protonated at low temperatures. At higher temperatures, we suggest ion pairing between anionic Glu222 and a proton hosted in WP1. Upon photoexcitation, these two recombine, while a second proton dissociates from the chromophore and either exits the protein using the short WW, or migrates along the GFP-barrel axis on the long WW. This mechanism reconciles the conflicting experimental and theoretical data on proton motion within GFP.

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1

Introduction

Proton mobility is crucial to protein function. Proton movement in proteins can be divided into two broad types. 1 In the first, a single proton transfer event occurs during the activity of the protein, for example acid-base reactions in enzymes. The second type of processes are those in which the proton moves inside a protein, sampling distances substantially larger than interatomic distances, and are better referred to as “proton transport” (PT). One family of proteins in which PT occurs are “proton pumps”. These transport protons across the cell or mitochondrion membrane. Well-known examples are bacteriorhodopsin 2 and cytochrome c oxidase, 3 which convert light or chemical energy, respectively, into a transmembranal proton gradient, which drives ATP synthesis. The most efficient mechanism for moving protons inside proteins is thought to involve a “proton wire” (PW). 4,5 This is a chain of hydrogen bonded (H-bonded) atoms on which the proton “moves” by breaking and forming contiguous hydrogen and covalent bonds. The moving element in the PW is the protonic charge rather than the actual proton, so a proton would attach itself at one end of the wire, and emerge on the other as a different proton. Inside proteins, PWs might be composed of oxygen, nitrogen and sulfur atoms of the amino acid residues, as well as oxygen atoms of internal water molecules. However, the different proton affinities of the different atoms along the PW may result in rather high barriers. Sometimes a chain of water molecules can form within a protein, and then we speak of a “water wire” (WW), which is a PW consisting exclusively from oxygen atoms of water molecules. The less heterogeneous proton carriers along the chain should facilitate PT. WWs were experimentally identified in photosystem II 6 , carbonic anhydrase II, 7 and other proteins 8–11 . Proton translocation along WWs differs from the Grotthuss mechanism which takes place in bulk water, 12 because in PWs protons move along preformed onedimensional chains of H-bonds. Yet short WWs are occasionally formed even in bulk water, 13 particularly as connecting elements between proton donors and acceptors. 14–16 In addition to transporting protons, WWs can transport electrons 17 or water molecules. For example, in Bovine Pancreatic Trypsin Inhibitor (BPTI), a WW serves for moving water molecules between the active site and the bulk in a so-called “aqueduct mechanism”. 18,19 An aqueduct was also found in Cytochrome P450, and suggested to act as a channel for both water and proton exchange with the protein exterior. 20 Several computational works, using quantum-mechanical (QM) or hy2


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brid QM/MM methods, demonstrated the utilization of proton 21–26 and water 7,27–31 wires as an effective mode for PT inside proteins. However, due to obvious computational limitations, these studies focused on a small number of PT events, usually near the active site. Currently, multi-step PT within proteins is not routinely simulated. The system of interest for the present work is the Green Fluorescent Protein (GFP) from Aqueoria victoria (wt-GFP), a soluble protein in which PT occurs at a critical stage during its activity. GFP is widely used in biological and medical research as a fluorescence marker. 32–35 It has a β-barrel structure consisting of 11 aligned β-strands. 36,37 (We define the “bottom” of the barrel as the base containing both termini of its amino acid sequence). The fluorescence occurs at a chromophore, located on an α-helix traversing the barrel axis. The chromophore forms after protein folding in an autocatalytic cyclization of three consecutive amino acid residues, Ser65, Tyr66, and Gly67, followed by dehydration and oxidation reactions, 38 see Figure S1 in the supporting information (SI). In GFP, fluorescence is triggered by photon absorption, initiating 39 an excited state proton transfer (ESPT) reaction. 40 In the ground electronic state, the chromophore is predominantly neutral (the A-state), absorbing at 395 nm. A weaker absorption peak at 475 nm is attributed to the anionic (deprotonated) form (B-state). 41–43 Emission (fluorescence) is red-shifted with respect to absorption, detected at 406 and 508 nm for the A and B states, respectively. Thus, as in solution, ESPT is characterized by a red-shifted B-band relative to the A-band. 40 Upon irradiation, a proton dissociates from the phenolic OH group of the excited chromophore (state A), leaving it in the excited anionic state (state B), which produces green fluorescence (at 508 nm) within about 10 ps. 39 The transition between states A and B appears to occur in two steps via an intermediate state I. 39,44,45 First, charge transfer occurs (A→I, 2.5 ps), then Glu222 rotates to the syn conformation (I→B, 10 ps), breaking its H-bond with Ser205. Hence state I has the charge state of B but the Glu222 conformational state of A. According to the generally accepted model, 44,46,47 the dissociated proton travels to the nearby (anionic) Glu222, protonating one of its carboxylic oxygen atoms. Infrared measurements of the (protonated) Glu222 carbonyl band at 1710 cm−1 indeed find a similar rise-time, sometimes together with a second time constant. 47–50 The distance between the phenolic oxygen atom of the chromophore (to be denoted OH-Cro66i ) and Glu222 is travi

Atom names follow the IUPAC-IUB rules, 51 also used by PDB and CHARMM format

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eled using a PW consisting of a water molecule and an OH group of Ser205 (see Figure 1). 44 This water molecule participating in the PW is central to GFP dynamics, therefore we termed it the “privileged water molecule” (PWM). Upon returning to the electronic ground state the proton is shuttled back to the chromophore along the same pathway. Several computational studies were devoted to the proton shuttling process along a model OH-Cro66→PWM→OG-Ser205→Glu222 pathway. 52–56 These suggest that a (near) concerted, multi-proton transfer takes place, reaching Glu222 in about 100 fs. This is considerably faster than either the 10 ps or 2.5 ps timescales suggested by experiment. 39 The above pathway between the chromophore and Glu222 may not be the only possible route taken by the photodissociated proton. Alternative pathways in the chromophore region were found in GFP mutants. 57,58 It was suggested that following the rotation of Thr203, a short pathway is formed, OH-Cro66→PWM→OG1-Thr203→O-His148, leading to the bulk via the backbone carbonyl of His148. 59 Some experimental work indeed implicated His148 as a gateway to the bulk solution. 60–63 In X-ray structures of several GFP-like fluorescent proteins (such as zRFP574, 64 TurboGFP, 65 KillerRed 66 , asFP595 67 and its mutant, the kindling fluorescent protein 68 ) a 3-water-molecule wire connecting the chromophore OH group with bulk water was observed. Molecular dynamics (MD) simulations revealed similar WWs in the T203V/S205A double mutant of GFP. 69 Our recent MD work 70 showed that the PWM in wt-GFP, which is H-bonded to the chromophore in all of the GFP X-ray structures, exchanges with bulk water through a hole in the β-barrel wall. Moreover, at times of water exchange, a complete WW occasionally traverses this hole. Additionally, several studies 59,71–73 located PWs along the main axis of the GFP barrel, connecting Glu222 to Glu5 at the bottom of the barrel. Interestingly, a WW traversing a similar route was observed in the X-ray structure of KillerRed. 66 There, several possible roles have been suggested for the WW, among which are carrier of reactive oxygen species, of electrons, or a proton wire (through which a proton may enter and stabilize the anionic chromophore). Connected to this wire, on the outside of the bottom surface of the barrel, is a negatively charged surface patch, 72 with properties akin to a “proton collecting antenna”. 74 This apparatus was suggested to attract protons from the bulk and funnel them further inside through the one-dimensional (1D) PW emanating from Glu5. 72 Possible experimental support for proton migration along these PWs (or files

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their WW counterparts) comes from transient fluorescence measurements from the excited neutral chromophore (prior to deprotonation). 71,75–77 After correcting for the excited-state lifetime of the chromophore, these data revealed a temperature (T ) dependent power-law behavior, switching from t−1/2 (at low T ) to t−3/2 (at high T ). The rise of the Glu222 carbonyl signal (at 1710 cm−1 ) is also non-exponential, but its exact functional form is difficult to determine. 50 A t−d/2 power-law generally suggests a reversible diffusion-influenced reaction between a pair of particles (“geminate” reaction) in d-dimensions. 40 Thus the low-T behavior might be indicative of proton diffusing along 1D PWs within the GFP, such as the long PW along the barrel axis leading toward Glu5. 59,71 However, fully 3D diffusion (leading to the t−3/2 behavior) might not be feasible in the protein interior. An alternative 1D model showing such behavior involves irreversible proton escape from the chromophore in competition with the reversible geminate recombination reaction. This suggests that a “hole” in the GFP barrel occasionally opens at the higher temperatures, allowing a short exit path out of the protein cage. The scenario in which the dissociated proton goes elsewhere besides Glu222 encountered serious criticism. 78 The timescale for proton dissociation from carboxylic acids is considerably slower than from hydronium, at least in liquid water (µs vs. ps). Thus a proton arriving at a glutamate is expected to stay there for the duration of the chromophore’s excited-state lifetime. An alternative origin for the power-law kinetics was ascribed to the rotameric states of Ser205 (that we investigate in more detail below). For one Ser205 rotamer the PW to Glu222 is intact and then A can convert to B. In the other state(s) the PW is broken, resulting in a chromophore state A0 that cannot convert to B. 78 This “gated” reaction can be described by extending the A → B kinetic scheme (exponential kinetics) to A0 A → B. The solution for the chemical rate equations for this scheme (by diagonalization of a 2 × 2 matrix), can lead to bi-exponential kinetics, but not to a power-law. How, then, might one explain the seemingly conflicting data in a consistent way? Missing from the discussion thus far was the possibility that the GFP kinetics involves more than one proton. For example, one proton may transit to Glu222 while the second diffuses along a PW reaching the chromophore site. The more detailed MD investigation initiated herein could shed some light on this fundamental question. The H-bonding pattern near the chromophore readjusts in the anionic B state. The Glu222-Ser205 H-bond is disrupted by the anti →syn transition of Glu222. 45 In addition, Thr203 rotates its OH group toward the 5



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chromophore, donating a H-bond to the negatively charged phenolic oxygen atom. 44,46,79,80 The rotation of Thr203 (and similarly for Ser205) occurs around its Cα –Cβ bond (see Figures 2 and 3), with the χ1 angle having three possible values, −60 , 60 , and 180 degrees (termed gauche−, gauche+ and trans or g-, g+, and t, respectively). However, in GFP Thr203 resides in an STQ sequence, which apparently reduces the conformational space to just two rotameric states (t and g-). 81 State t is usually observed in state A (of the chromophore), whereas state g- is observed in state B. The restriction of Thr203 to two rotameric states extends to other fluorescing proteins with GFP-like structures. 82 This relation however is not exclusive, because in the highest resolution X-ray structure of the wild-type GFP (wt-GFP, PDB code 2WUR 72 ) Thr203 exists in the g- state despite being in the A-state (neutral chromophore, intact PW between OG-Ser205 and Glu222). The g-/neutral chromophore combination was recently found in 41 % of the 71 fluorescent proteins that had a threonine residue in a position corresponding to Thr203 in Aequorea victoria GFP. 82 In addition, a recent QM and QM/MM study 45 that characterized the structures and energies of the GFP states (A, B, and I), showed that there are more than two stable charge/structure combinations, and that in state B the g- state of Thr203 is not the lowest in energy. The study did not, however, examine the probability of state A having Thr203 in state g-. A primary source for protein structure, and hence for PW structure, are X-ray measurements on protein crystals near 0 K. These reveal invaluable information without which it is difficult to study protein structure and function. Yet, the X-ray results may be misleading in at least two respects. Firstly, these structures are near 0 K and are static. Biology thrives near room temperature, where there is extensive dynamics taking place on all distance and timescales. Amino acid sidechains move rapidly, backbone conformational changes are more time consuming, water molecules move in and out of the protein and H-bonds break and reform. It is not possible to cope with this complexity without theoretical tools, particularly MD, which has become as indispensable as X-ray for a wide array of biological applications. Secondly, protein crystals may be partially dehydrated as compared to the protein in aqueous solutions at room temperature. Indeed, we see in our simulation how the number of internalized water molecules gradually grows as we subject an initial X-ray structure to Newton’s laws. Thus, while PWs seen in X-ray structures may provide an instructive overall sketch for how these “wires” are constructed, some missing pivotal water molecules may be seen only with MD. 6


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Several groups have conducted MD simulations of GFP and its mutants. 70,73,78,83,84 Here we have undertaken a detailed MD study of the PW dynamics in the wt-GFP A-state chromophore. We generated a > 300 ns trajectory, which is sufficiently long for sampling configurations that are not accessible in the X-ray structures. Specifically, we determine the characteristic PWs for the different rotameric states of Ser205 (fast rotation) and Thr203 (slow rotation), a total of 9 states. Contrary to the X-ray results, we find that water insertion occurs between Ser205 and Glu222, extending the Cro66 to Glu222 PW by one unit. We apply a random walk search for other PWs emanating from the OH-Cro66 atom, finding that a proton originating from the Cro66 hydroxyl group can escape to the bulk using several exit points in the chromophore vicinity, most notably a direct WW connecting the PWM via two additional water molecules to the bulk. A “water pool” (WP1) in the volume between Ser72 and Glu222 supplies the inserted water molecules, and connects via a long “axial WW” to the barrel bottom. These and other findings are described below.

Figure 1: Schematic drawing presenting H-bonds in the chromophore (in green) region for (A) state A (neutral chromophore, anionic Glu222) and (B) state B (anionic chromophore, protonated Glu222) according to the generally accepted model 44,46,47 for PT in the GFP chromophore region. Dashed lines represent H-bonds.

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Figure 2: The χ1 angle of threonine/serine residues. For threonine X is CH3 , for serine X is a hydrogen atom.

Figure 3: The three rotamers of threonine g-, t, and g+, shown in a 3D projection (top row), and in a Newman projection (bottom row). Rotation is around the CA–CB (Cα –Cβ ) bond (perpendicular to the viewer, not visible in the figure), the measured dihedral angle is OG1–CB–CA–C.

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2 2.1

Methods MD simulations

Classical MD simulations for GFP in water were performed with the NAMD 2.8 program, 85 using the CHARMM27 force field 86 with the TIP3P water model. 87 Force field parameters for the chromophore were taken from Reuter et al., 88 see Tables S1–S5 in the SI. A crystal structure with PDB entry 2WUR 72 (resolution of 0.90 ˚ A) was used as an initial structure. In this structure, which is the highest resolution X-ray structure of wt-GFP to date, some hydrogen atoms of protein residues are visible. Several VMD plugins were utilized to set up the system. Hydrogen atoms of water molecules were added using the Psfgen plugin (ver. 1.4.7). The Solvate plugin (ver. 1.4) was used to solvate the protein in a cubic water box with 10 ˚ A padding in each dimension (with 10 520 water molecules added). The Autoionize plugin (ver. 1.3) was used to neutralize the electric charge of the molecule by adding Cl− and Na+ ions, reaching ionic strength of 0.05 M. The total number of atoms in the simulation was 35 174. Histidine protonation states were taken from the 2WUR PDB file. These were set 72 on the basis of the calculated pKa values using the PDB2PQR software. 89,90 Because the pH was 8.0 in the crystal, occupancies of acidic protons were set to 0.0 for pKa < 7.0, 1.0 for pKa > 9.0, and intermediate values otherwise, as detailed in ref 72. In all histidines only atom ND1 is protonated, excepting His25, in which both ND1 and NE2 are protonated. Because at 0.9 ˚ A resolution many hydrogen atoms become visible, 91 this conclusion could be verified directly from the Fo − Fc hydrogen omit map, see histidines 148 and 181 in Figures 4 and 5 (respectively) of ref 72. The sidechains of several residues show double occupancy in the X-ray structure. For these atoms, the conformation with the larger occupancy was used. All but one (Leu42) doubly occupied amino acid sidechains point to the solvent. Leu42 resides above the chromophore plane, away from the active site, thus not expected to participate in the currently studied dynamics. Two internal water molecules, Wat2109 and 2308, were also doubly occupied. These are water molecules from WP1, discussed in Sec. 3.3.5 below. The short–range electrostatic and van der Waals interactions were gradually switched off between 10–12 ˚ A. The long–range electrostatic interactions were calculated using the particle mesh Ewald method. 92 In accordance with the use of a rigid water model, hydrogen–oxygen and hydrogen–hydrogen distances in water molecules were constrained using the SHAKE algo-

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rithm. 93 The integration time step was 1 fs, with coordinates saved every 500 fs for analysis. The temperature during the simulation was kept constant using the Langevin thermostat with a damping coefficient of 5 ps−1 . The pressure was maintained at 1 atm using the Langevin barostat 94 with a piston period of 200 fs and damping time of 50 fs. The simulation procedure was performed as follows. After energy minimization of the X-ray structure using the conjugate gradient algorithm, the system was propagated under constant volume and temperature for 100 ps for thermal equilibration to a target temperature of 300 K. Then it was continued under constant temperature and pressure (NPT ensemble) for 1 ns. During the last two stages, all non-hydrogen atoms were harmonically constrained to their minimized coordinates with a force constant of 20 kcal/mol/˚ A2 , after which the constraints were removed and the system was propagated under constant temperature and pressure for 307 ns. The first 1 ns was used for equilibration, the total simulation time used for analysis was 306 ns. Superposed X-ray, minimized and MD structures are shown in Figure S3 of the SI.

2.2

Analysis of results from simulation

Data visualization was performed using VMD 1.9. 95 Analysis scripts were written in the Tcl 8.4.1 scripting language embedded into VMD and extended to include VMD commands. 2.2.1

Identifying proton wires connecting two atoms

Two atoms were considered as having a PW between them whenever a continuous chain of H-bonded atoms connected them. Several criteria for determining H-bonding between atoms can be found in the literature. 96–100 Here, geometrical (distance-angle) criteria were used as follows. 101 Atoms A1 and A2 were considered directly H-bonded if the distance between them was under 3.5 ˚ A, one of them carried a hydrogen atom, and the smallest A1 –H–A2 angle was larger than 120°. Atoms that may participate in a PW included all oxygen, nitrogen, and sulfur atoms of the protein (sidechain and backbone), as well as oxygen atoms of water molecules. The search of a PW between atoms A1 and A2 was carried out as follows (somewhat similar algorithms were previously implemented for detecting electron transfer pathways in proteins 102,103 ). Starting from A1 , we collect all the atoms that are H-bonded to it. This defines the first “layer” in the PW search. If one of them is A2 , there is a PW between A1 and A2

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and the search is terminated. If not, the search continues by collecting all the newly encountered atoms that H-bond to at least one of the first layer atoms. H-bonded layers are kept being added until either a layer in which A2 appears is reached, or until a maximally allowed number of layers is reached (in which case there is no PW). In order to detect the composition of the PW we follow back the layers. Starting from A2 , in each layer we collect all the atoms that are H-bonded to the previous layer, until A1 is reached. In this way, all possible PWs between A1 and A2 are detected. Sidechain carboxyl oxygens of glutamate (and aspartate) residues are symmetric so that each of the two atoms (OE1 and OE2 for glutamate) can participate in PWs interchangeably. Depending on the surrounding, the sidechain can easily rotate and the atoms can exchange roles during the simulation, therefore PWs involving Glu222 were searched for both of the carboxyl atoms and the shortest PW of the two was selected. We define the length of the PW as the number of atoms mediating the two end atoms. In this nomenclature, a direct A1 · · · A2 H-bond involves a PW of length 0, A1 · · · W1 · · · A2 is a PW of length 1, etc. The cutoff length was defined as the X-ray length of the PW plus 2 or 3 (depending on the specific PW). The water fraction of a PW is the fraction of water oxygen atoms out of all atoms participating in the PW, not including the end atoms. For convenience, we will refer to the individual PWs by the residue numbers of their end atoms. For example 66–205 will denote the PW between OH-Cro66 and OG-Ser205. 2.2.2

Proton wires emanating from a single atom

PWs originating from a single atom were searched for using a random walk algorithm. This search, with no preselected end atom, propagates at each step to a H-bonded atom selected randomly from several possible candidates. Starting from an atom A0 , we consider all atoms that are H-bonded to it, selecting one of them randomly as atom A1 , the next atom in the PW. In the next step, we consider all atoms H-bonded to A1 , excluding A0 . The process continues until the maximal number of steps (n) or an atom with no additional H-bonding partners are reached, in the latter case the PW is shorter than the maximal length. Multiple starting times along the trajectory are selected randomly, and for each one the PW search is initiated multiple times. A flowchart illustrating the algorithm is presented in Figure S2 of the SI. The time separation between adjacent neighbor selection steps, (∆t), is 11



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also a tunable parameter, such that ∆t = 0 finds PWs in which all H-bonds exist simultaneously, whereas for ∆t = 1 ps the time between selection steps is 1 ps. Generally, ∆t > 0 produces longer PWs than ∆t = 0, because the elapsed time between selection steps allows the H-bond of the last move to break and be replaced with a H-bond to a new neighbor, increasing the number of possibilities for extending the wire. If ∆t is a typical proton hopping time, the wire selected also mimics more realistically the wire that a moving proton encounters, which can reconfigure between hopping steps. Here a value of ∆t = 0.5 ps was utilized. The starting atom for the random walk search was OH-Cro66 (except for Sec. 3.3.6, where OE2-Glu222 was the starting atom). Values for the tunable parameters were selected after testing several values and are as follows. The number of steps was n = 15 (not including the starting atom), because a larger value simply continues the PW into the bulk. A total of 5100 different starting times (t0 ) were selected randomly from the 306 ns trajectory, (100 t0 ’s per each 6 ns block of trajectory), with 100 new searches for each t0 . This search provided a total of 510 000 PWs, some of which may be identical. Increasing the density of sampling by increasing values for the last two parameters did not result in new PWs being discovered.

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3

Results and discussion

In this work, we have monitored the PWs emanating from the proton release site, OH-Cro66, in the wt-GFP A-state, as a function of the rotameric states of Thr203 and Ser205. This includes the “canonical” 66–222 PW, 44 as well as previously unreported (or rarely reported) PWs: (a) the water-elongated 66–222 wire (Figure 13); (b) the Ser205 bypass (Figures 11B and 12); (c) “direct exit” PWs leading from the chromophore directly to the bulk, such as the “hole in the barrel” WW (Figure 9), and (d) pathways connecting the chromophore with the barrel’s bottom (Figure 17). Thus MD reveals many more pathways than seen in X-ray structures. These pathways will now be presented in detail.

3.1

X-ray data

The wt-GFP X-ray structure 2WUR (0.9 ˚ A resolution) 72 was the starting configuration for the dynamics. We therefore start by briefly discussing some characteristics of this structure relevant for the analysis below. Other Xray structures, 1EME (2.5 ˚ A resolution) 46 and 1EMB (2.13 ˚ A resolution) 44 , were used as starting points for previously calculated trajectories, 70 which are discussed in the SI. Thr203 can exist in three rotamric states (Figure 3). However, only two of them are usually observed in GFP X-ray structures: 81 In the A-state (neutral chromophore) Thr203 is mostly in the t state (e.g., Figure 4B), rotating to g- in the B-state (anionic chromophore). In the 2WUR structure used herein, Thr203 is in the less common g- state although its chromophore is neutral (Figure 4A). Although the Thr203 OH group is within H-bonding distance to the chromopohore, it points its H atom toward the PWM. While this layout is considered more common to the B-state, further away from the chromophore the H-bond between OG-Ser205 and the carboxyl oxygen of Glu222 also exists, a characteristic of the A-state. Nevertheless, the directionality of the 66-222 PW is reversed. The hydrogen atom of the Ser205 hydroxyl group, which is visible in the 2WUR structure, points toward the PWM (Figure 4 in ref 72) rather than to Glu222, as postulated for the A-state. This could indicate a protonated Glu222 (Sec. 5.2), rather than a deprotonated state of the chromophore. While a mixture of the two Tyr66 protonation states is possible, the dominant species at pH=8.0 (the pH of the 2WUR crystal) should have a protonated chromophore. In denatured wt-GFP, the pKa (from the pH of the absorption isosbestic point) is 8.1, 41 close to the 2WUR crystal pH value. However, the

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folded protein does not reach this point until close to the denaturing pH of about 12. 42 This suggests that the ground-state chromophore is protected (by the beta-barrel) from deprotonation even at rather high solution pH. The present simulations are for the canonical A-state, with protonated Tyr66 and deprotonated Glu222. In this case we observe, already during the energy minimization step, that the Ser205 OH group rotates, breaking its H-bond with the PWM and donating it to Glu222 (see Figure S3A in the SI), thus forming the “canonical” PW suggested in the literature. 44

Figure 4: H-bonds (dashed black lines) in the chromophore region for the minimized X-ray PDB structures 2WUR and 1EME. Distances (in ˚ A) between the atoms sharing the H atom are presented next to the lines. Dashed red lines connect atoms that are within H-bonding distance, but do not have a proper H-bond angle. See also Figure S3 in the SI for a 3D superposition of the chromophore region.

3.2

Thr203 and Ser205 rotameric states

The simulations show a richer sequence of events than what could be deduced from the X-ray structures. While these have Thr203 in either its t or g- states, 81 and Ser205 always in the t state, during the simulations both Thr203 and Ser205 sidechains sample all three states g-, g+, and t. For Thr203, the rotameric dynamics of the χ1 angle is slow, with each state reached once or twice during the 306 ns simulation (see Figure 5D). Thr203 spends 112, 99, and 95 ns in g-, g+, and t, respectively, dividing its time roughly equally between the three states. Due to its smaller sidechain, Ser205 exhibits faster rotameric dynamics than Thr203 (Figure 5B), with an average of 0.9 ns between adjacent transitions. Rotations of Ser205 occur more frequently in the Thr203 g- state 14


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than in its t state, where the bulky methyl group is closer to the Ser205 side chain (Figure 5C), blocking its rotation. The three Ser205 rotamers are found with nearly equal probabilities (0.38, 0.25, and 0.37 for g-, g+, and t, respectively), quite in contrast to the exclusive t state seen in the X-ray structures. While the fast isomerization of Ser205 was observed in a previous 60 ns simulation, 78 that simulation was too short to reveal any Thr203 rotameric dynamics. Additionally, Ser205 isomerized between just two (unspecified) rotameric states. Thus only two joint Ser205/Thr203 states were sampled, whereas here we report all nine of them. Figure 6 presents snapshots of the nine joint S205/T203 rotameric states, whereas Table 1 lists the occupation fractions, Pij , for these states. Here i = 1, 2, 3 and j = 1, 2, 3 are the g-, g+ and t rotameric states of Ser205 and Thr203, respectively. P The overall occupancies for the Ser205 states in the last column, pi = j Pij , are very different from the t state dominance seen in the X-ray data. This likely results from the insertion of a water molecule between Ser205 and Glu222 described below. P The overall occupancies for the Thr203 states in the bottom row, pj = i Pij , are nearly equal for the 3 states, though the pj values may be inaccurate due to the slow Thr203 rotameric dynamics. The g+ rotamer of Thr203 is hardly ever found in the X-ray structures of wt-GFP, 81 although it has high occupation in the X-ray structures of other proteins. 104 This was attributed to the local effect of the Thr203 neighboring amino acids along the GFP backbone, and specifically to its occurrence in a STQ sequence. 81 Our observation that Thr203 state g+ has regained its prominence in the 300 K dynamics therefore likely reflects non-local conformational changes (away from the STQ triad) that occur prior to the transition to g+. We have therefore inspected the possible role of backbone movements in readjusting the Thr203 sidechain location. Table 2 presents trajectory-averaged distances between pairs of residues in opposing strands 7 and 10 (cf. Figure 2 of Ref. 70), for the three Thr203 rotameric states. It is seen that state g+ is reached once the 144-207 distance increases significantly, whereas the other two distances slightly decrease. The Asn144 residue, in turn, is located at the end of strand 7, close to the large “129–142 loop”, which caps the GFP barrel. Its motion was suggested to assist in opening the “hole in the barrel” between strands 7 and 10, through which the PWM exchanges with bulk water. 70 Indeed, the RMSD of residues 129-142 shows a sudden jump slightly before the transition of Thr203 to its g+ state (∼180 ns). The RMSD profile, as well as distances between residues on strands 7 and 10 are presented in Figure S4 in the SI. 15



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The increased motion of the backbone in this region can readjust the interactions of the Thr203 sidechain so as to stabilize the g+ rotamer. One such possible consequence of the backbone motion is that in state g+ Thr203 is closer to Glu222, allowing the formation of a H-bond between OG1-Thr203 and the carboxylic oxygen of Glu222 (Figure 7B). In the minimized structure (before backbone movement, Figure 7A) the distance between the closest Thr203 and Glu222 atoms (HB-Thr203 and OE2-Glu222) is 3.91 ˚ A, not allowing a H-bond to form even if the sidechain was rotated in such a way that OG1-Thr203 was the closest to Glu222. Movie S1 presents a 100 fs segment from the MD trajectory (at 209 ns) showing Thr203 rotation and (OG1-T203)-(OE2-E222) H-bond formation.

16


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Figure 5: A) (top) Length of the 66–205 PW over time, t. Times with no data have no PW. For clarity of vision data were smoothed as follows. At each t, data were collected from t − 10 to t + 10 ps. If for > 50 % of the time a PW (of any allowed length) exists, t was considered as having a PW, for which the length was calculated as the average of the lengths of the existing PWs within the time range. If a PW did not exist for > 50 % of the time, the point was considered as not having a PW. B) χ1 dihedral angle as a function of t for Ser205. C) Distance between CG2-Thr203 and CB-Ser205. D) χ1 dihedral angle as a function of t for Thr203.

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Figure 6: Snapshots along the simulation in which Ser205 and Thr203 are in different rotameric states. The chromophore and the PWM are also shown in the figure. Dihedral angle states for Ser205 and Thr203 are written in the bottom right corner of each panel and presented in matrix notation corresponding to the 3 × 3 matrix in Table 1. Dotted blue lines denote H-bonds. Snapshots were taken at 10.8, 15.2, 2.3, 280.0, 247.0, 258.9, 190.0, 170.0 and 123.2 ns for Figures A–I. (C) depicts the rotameric states of the 2WUR X-ray structure. (I) depicts the rotameric state of the 1EMB and the majority of GFP X-ray structures. 18


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Table 1: Occupancya of joint Thr203 and Ser205 rotameric states Ser205/Thr203 gg+ t Thr203 a

b c

g+ 0.125 0.167 0.031 0.323

t Ser205 0.135 0.384 0.028 0.249 0.146c 0.367 0.310 1

Fraction of time, Pij , during the simulation, spent in Ser205 state i and Thr203 state j. 2WUR X-ray structure. 1EMB X-ray structure.

Table 2: residues T203 state gg+ t a

g0.123 0.054 0.190b 0.367

Average distance between β-sheet Distance, ˚ A N144-L207a N146-S205a H148-T203a 7.62 8.79 7.37

6.12 5.80 5.96

3.76 2.99 3.12

Distances measured between backbone oxygen atom of one residue to the backbone nitrogen atom of the other residue and vice versa. The two distances were averaged.

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Figure 7: Snapshots taken for (A) the minimized 2WUR structure and (B) during the simulation at 228 ns, showing the chromophore, Thr203, and Glu222. The rotameric state of Thr203 is g- for (A) and g+ for (B). Dashed blue lines connecting atoms from Thr203 and Glu222 for which distances are written in ˚ A next to the line.

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3.3

PWs emanating from OH-Cro66

Figure 8 presents the most common H-bonds appearing in PWs emanating from the chromophore, as found by the random walk search from OH-Cro66 over the course of the simulation. The data is presented separately for the three Thr203 rotameric states. H-bonds shown in the figure constitute > 3 % of all H-bonds for the given state. In all three states the atoms comprising the X-ray 66–222 PW appear, as well as additional atoms found only with the random walk search. The PW from OH-Cro66 to OG-Ser205 appears in its X-ray form for all three states. The dependence of the PW probability on the Thr203 and Ser205 states will be discussed in Section 3.3.1. In Ser205 states g+ and t (Figure 8B and C) the PWM (marked as W0-66) is H-bonded also to O-ser205, which in turn, has an exit to the bulk. Exit pathways will be discussed in Section 3.3.2. In state g+, OH-Cro66 and Glu222 are connected by a direct WW, bypassing Ser205. Such alternative 66–222 PWs will be discussed in Section 3.3.3. In the GFP X-ray structures, a direct H-bond between Ser205 to Glu222 is always observed. In contrast, during the simulation, for all three states, a water molecule is seen mediating Ser205 and Glu222. This previously unreported water insertion, and its coupling to water dynamics in WP1, will be discussed in Section 3.3.4.

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Figure 8: PWs found with a random walk search originating from OH-Cro66, shown separately for Thr203 states g-, g+, and t (panels A, B, and C, respectively). Each H-bond presented (solid lines) constitutes > 3 % of all H-bonds found in the PW search, with a thickness corresponding to its frequency of appearance. Atom names are written in sizes proportional to their frequency of appearance in individual H-bonds, major atoms are colored for clarity. Water molecules were identified and named according to the preceding protein atom on paths emanating from OH-Cro66. WWs of length > 5 were treated as exiting to the bulk and are marked by “bulk” (pathway cut short for clarity). Dashed lines represent bonds that constitute < 3 % of the H-bonds found but connect to a more frequent H-bond farther down the path. 22


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3.3.1

The OH-Cro66→PWM→OG-Ser205 PW

We have investigated the 66–205 PW independently from the 66–222 PW, because Ser205 does not participate in all of the 66–222 PWs. We find that the 66–205 PW exists during 65 % of the simulation time, and then it retains its “canonical” form, 44 OH-Cro66 → PWM → OG-Ser205, which differs in Ser205 OH group orientation from the 2WUR X-ray structure (see Sec. 3.1). For this PW we find an average length of 1.18 and a water fraction of 0.972 (both calculated as an average over all frames in which the PW exists). Thus OH-Cro66 and Ser205 are bridged exclusively by water molecules (hence the water fraction of 1), mostly a single one (the PWM). Occasionally (as in Figures 6E and F) they are bridged by two water molecules (and hence the average PW length is > 1.0). The rotameric state of Ser205, which is directly involved in the PW, is expected to greatly influence the existence of the PW. Thr203, although not directly involved in it, lies in proximity to the wire and its rotameric state is also expected to influence the PW. Hence we checked the probabilities for having this PW for given rotameric states of Ser205 and Thr203. Table 3 (see also Figure 5) presents the conditional probabilities, Qij , for PW formation in each of the 9 combined states (as defined in Section 3.2). The overall PW probabilities for the three Ser205 states (Qi , rightmost column) and the three Thr203 states (Qj , bottom row) are given by: Qi = 1 P 1 P j Pij Qij and Qj = pj i Pij Qij , respectively (with pi and Pij from pi Table 1, above). Thus Qi is an average of Qij over j, and inversely, Qj is an average of Qij over i. This is reflected in their values shown in Table 3. Because Table 3 presents conditional probabilities (given that the system is in a specific Ser205/Thr203 state), the significance of the PWs listed therein should be assessed with the help of Table 1, which lists the probabilities for these conformational states. For instance, while in state t/g+ the 66-205 PW has a relatively high fraction (Table 3, 0.578) due to its elongation by an additional bridging water, the overall fraction of this state is low (Table 1, 0.031). Therefore this specific case occurs for very short times and is not representative of the dynamics observed. Figure 6C represents the 2WUR X-ray structure t/g- state, with Ser205 turned toward Glu222 (not depicted in the figure), completing the “canonical” PW (OH-Cro66 → PWM → OG-Ser205 → OE2-Glu222). There is a high probability for observing this PW in this rotameric state. In comparison, state t/t (Figure 6I), which supposedly corresponds to the majority of the GFP crystal structures, shows a surprisingly low probability for having a 66–205 PW (0.070, Table 3). Inspection of our trajectory shows that this 23



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state is not identical to the t/t state encountered in the 1EMB structure. The rotation of Ser205 from g- to t (to form the t/t state), at 122 ns, is initiated by an earlier configuration change of Glu222, at 112 ns, which tilts its sidechain downward, away from the chromophore region (Movie S2 in the SI). In order to maintain the 205–222 PW, Ser205 moves with its sidechain toward Glu222 and at the same time, rotates to state t (Movie S3 in the SI). In this configuration, the distance between OG-Ser205 and OH-Cro66 is too far to form the original PW. When Ser205 is in the state g+ (Figures 6B, E, and H) it turns away from the PWM and the H-bond connecting them is broken. This results in the low PW probabilities for the middle row of Table 3, except that in the g+/g+ state (Figure 6E) an alternate PW constructed of 2 water molecules is observed. However, the probabilities for the g+/g- and g+/t states (for which the 66–205 PW rarely exist) are themselves small (Table 1), so that the overall probability for the 66-205 wire remains high.

24


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Table 3: 66–205 PW probabilitiessa for different Thr203 and Ser205 rotameric states Ser205/Thr203 gg+ t Thr203

g0.888 0.034 0.913b 0.776

g+ 0.899 0.546c 0.578d 0.686

t 0.954 0.057 0.070 0.455

Ser205 0.915 0.380 0.549 0.648

a

For PWs of all allowed lengths. For all states except g+/g+ and t/g+ (see notes c,d ) the dominant length is 1, as in the X-ray structures. b 2WUR X-ray state. c Average length of PW is 1.70, probability for PW of length 1 is 0.260. d Average length of PW is 2.24, probability for PW of length 1 is 0.025.

3.3.2

Water wire and other exit pathways from OH-Cro66 to the bulk

A PW ending in the bulk is of infinite length because, evidently, in the bulk a water molecule always has several H-bonding partners. In our random walk search, all PWs ending in the bulk have the maximally allowed length of 15. From all PWs of this length, only those ending in a water molecule were considered as leading to the bulk. The last protein atom in such a PW was regarded as its exit point. When the exit point is a protein atom we speak of an “exiting PW”, whereas when it is OH-Cro66 itself it is an “exiting WW”. The fraction of exit pathways out of all PWs emanating from the chromophore was largest for the Thr203 g+ state (19 %), followed by state t (13 %) and finally g- (8 %) (the overall fraction is 13 %). (i) Exiting PWs. As calculated by the random walk search, an exit to the bulk is the main competing pathway to the conventional 66–222 route. We have calculated the ratios “222:bulk”, of random walk PWs reaching Glu222 (either one of its two carboxylic atoms) vs. those reaching the bulk (any pathway leading to bulk, expect those involving Glu222). We find this ratio to be surprisingly small, with 1:0.68 and 1:0.87 for Thr203 g- and g+, respectively. For state t, this ratio becomes even smaller than unity (1:2.35), perhaps because steric hindrance with the Thr203 methyl group pushes aside Ser205 thus interfering with the pathway to Glu222 (compare 25



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Sec. 3.2). For rotamers g+ and t of Thr203, O-Ser205 is the most common exit point, and the path leading to the bulk is shown in Figure 8B and C (OH-Cro66→PWM→O-Ser205→bulk). For the g- state, O-Tyr145 is the most common exit point and the exit path is elongated by an additional water molecule after the PWM (OH-Cro66→PWM→Wat→O-Tyr145→bulk). Interestingly, the exit point through O-His148, previously deduced 59 from the B-like structure of the S65T mutant (Thr203 in its g- state), is not seen in our A-state simulations, though it was observed in Fig. 5D of Ref. 73. We have checked this with several of our methods (e.g., random walk and frame-by-frame searches), and all of them are consistent regarding the observation that there is no exit from OH-Cro66 through O-H148. (ii) Exiting WWs. An exiting WW occurs when OH-Cro66 itself serves as an exit point. The “abnormal” Thr203 g+ state has the largest fraction of exiting WWs (17.2 % of all exit paths and 3.2 % of all PWs produced by the random walk search for the g+ state). State t has the smallest fraction of WWs (7.8 % of exit paths and 1.0 % overall), perhaps due to the competing O-Ser205 exiting PW, which dominates state t. Overall, 14 % of all exit paths are WWs. Figure 9 presents a snapshot taken at 4130 ps showing a WW leading from OH-Cro66 to the bulk. The water molecules along this wire H-bond to protein atoms in an order matching the “binding–layers” for PWM exchange through the “hole in the barrel” discussed in our previous work. 70 While this exiting WW has not been observed in any X-ray structure of wt-GFP, we note that the X-ray structures of several GFP-like fluorescing proteins (such as zRFP574, 64 TurboGFP, 65 asFP595, 67 and KillerRed 66 ) do show a similar WW. Apparently, in GFP one needs to go to room temperature and above to see the PWM exchanging with bulk water and hence also the Cro-to-bulk WW. 70 In order to systematically describe the path of the WWs from the random walk search we performed the following analysis. A water molecule in a WW was assigned an index I indicating its location in the WW starting from 1 (assigned to the PWM). All protein atoms that H-bond to water molecules in WWs were collected and assigned the indices I of the water molecules to which they H-bond. For each unique protein atom, indices I from all its H-bonds with WW atoms were averaged and a unique Iavg index was thus assigned to that atom. Hence Iavg represents the average location of the protein atom in the WW (as a neighbor). Figure 10 (compare with Figure 7 in Ref. 70) presents common protein atoms in contact with the WW with Iavg values smaller than 4.0. All the 26


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atoms presented appear in the two internal binding pockets as defined in Ref. 70 (Section 3.2). The three protein atoms that were assigned to the first binding layer in Ref. 70 (OG1-Thr203, OG-Ser205, and O-Asn146) have Iavg values of 1.01–1.36 showing that here too, they are H-bonded directly to the PWM. For a full list of Iavg and probabilities of appearance near WWs, see Table S6 in the SI. A frame-by-frame search for WWs connecting OH-Cro66 to the bulk was performed using the algorithm described in Section 2.2.1, limiting the participating atoms to water oxygens atoms, and defining the end atom as a water oxygen atom that does not have any protein atoms within a radius of 3.5 ˚ A. It was found that WWs exist for 71, 64, and 40 % of the simulation time for Thr203 states g-, g+, and t, respectively (overall: 59 %).

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Figure 9: An exiting WW connecting OH-Cro66 to bulk water, detected with the random walk search at 4130 ps. Dashed blue lines denote H-bonds. Protein atoms H-bonded to water molecules in the WW are shown, with their names written next to them.

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Figure 10: Protein atoms H-bonding to water molecules from the WWs found in a random walk PW search from OH-Cro66. Atoms presented are with Iavg < 4 and participate in > 2 % of all H-bonds to WW water molecules. Atoms appear in gradient colors, according to their Iavg value. Note how all the residues H-bonding to the exiting WWs are from strands 7 or 10.

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3.3.3

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PW between Cro66 and Glu222, bypassing Ser205

The 205–222 PW exists during most of the simulation, therefore the probability for observing the 66–222 PW is determined mainly by the probability of the less frequent 66–205 PW. Indeed, the direct search yielded a probability of 0.69 for the 66–222 PW, just slightly larger than that of the 66–205 PW (which is 0.65). This suggests that alternative 66–222 PWs exist. Searching for 66–222 PWs bypassing Ser205 (by not allowing PWs including Ser205), we find that such PWs exist to a significant degree only in the “abnormal” Thr203 g+ state (the PW probabilities are 0.103, 0.691, and 0.108 for Thr203 rotamers g-, g+, and t, respectively). In order to bypass Ser205, the PW must pass under the Ser205–Glu222 plane. In states g- and t the bulky sidechains of Thr203 (OH and CH3 groups for g- and t, respectively) block the first (in state g-) and second (in state t) water molecules in the alternative PW (Figure 11A and C). Within the Thr203 g+ state, the rotameric state of Ser205 affects the form of the PW. For Ser205 states g- and g+ the most common PWs are of length 2:

OH-Cro66 → W → W → OE2-Glu222 OH-Cro66 ← W → W → OE2-Glu222

(1)

for states g- (Figure 11B) and g+ (Figure 12A), respectively. However, for the Ser205 t state the most common PW is of length 3, OH-Cro66 ← W ← W ← W → OE2-Glu222,

(2)

see Figure 12B. Note how, for Ser205 states g+ and t, the direction of some of the H-bonds is inverted. We also note that in ∼30 % of the 66–222 PWs of length 3 in the Thr203 g+ state, OG1-Thr203 replaces the third water molecule in the above wire.

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Figure 11: Snapshots from the simulation showing the positions of the chromophore, Thr203, Ser205, and Glu222 for Thr203 states g- (A), g+ (B), and t (C). Snapshots taken at 96, 210, and 165 ns for (A), (B), and (C), respectively. Only in (B) there is a Ser205-bypassing PW between OH-Cro66 and OE2-Glu222 (with Ser205 in the g- state). Also see Movies S4-S6 presenting a rotational view of the scene.

Figure 12: Snapshots from the simulation showing the positions of the chromophore, Thr203, Ser205, and Glu222 for Ser205 states g+ (A) and t (B) (Thr203 is in state g+ for both panels). Snapshots taken at 228 and 225 ns for (A) and (B), respectively.

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3.3.4

The Ser205→Glu222 segment and water insertion

The 205–222 PW exists for most (92 %) of the simulation time. However, its length (which is 0 in the X-ray structure) averages to almost 1 during the simulation. A water fraction of 0.844 suggests insertion of one mediating water molecule. Indeed, inspection of the trajectory shows that very early into the simulation (4.6 ns, Figures 13, 14, and Figure S3B in the SI) a water molecule inserts between Ser205 and Glu222 and, excluding the time interval 122–164 ns, the PW is always mediated by a water molecule. This is unexpected from any of the X-ray structural models of wt-GFP, 44 which do not show this intervening water molecule. In a recent MD simulations of wt-GFP 78 starting from the 1GFL 37 X-ray structure, water insertion was not identified. However, in another recent simulation 73 that started (as here) from the 2WUR X-ray structure, 72 the authors have identified a hydration site between Ser205 and Glu222 that can bridge the H-bond between them (see Figure 5D in ref 73). Hence we followed this water insertion process in more detail. Figure 14 shows the 205–222 PW length and the dihedral angle of Ser205 as a function of time t. Water insertion occurs while Ser205 is in the t state, with its OH group pointing toward OE2-Glu222. Shortly after this, Ser205 rotates to the g- state, distancing itself from Glu222. The second period in which the PW is not mediated by a water molecule also has Ser205 in its t state, but after water insertion (at 180 ns), it rotates to the g+ state. Once Thr203 is in the g+ state (from 209 ns onward), the length of the PW sometimes exceeds 1, rising (momentarily) up to 3. Indeed, Table 4 shows that the average 205-222 PW length is somewhat larger in the g+ state. The table also shows the PWM exchange rate through the “hole in the barrel”, which we have studied in detail before. 70 The water exchange rate is noticeably larger in the g+ state, and some of the entering water molecules likely accommodate the 205-222 wire. A detailed analysis of water insertion events, correlated with Ser205/Thr203 rotameric states is presented in the SI (Section S2.3, Figures S5–S7, and Table S7). The higher temperature trajectories discussed there allow for more water insertion events and Thr203 conformation changes (whereas only 4 occur in our room temperature trajectory, see Figure 5D). A central observation in the SI is that the first water insertion event occurs when Thr203 is in the g- state. This distances the hydrophobic Thr203 methyl group from Ser205 (Figure 5C), allowing it more conformational flexibility (Figure 5B), and allowing a polar water molecule to approach. Previous simulations have not observed the Thr203 g- state, and this is 32


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possibly why the water insertion process was not seen before.

Figure 13: Snapshots taken before (left, 4517 ps) and after (right, 4679 ps) Wat2104 is inserted between OG-Ser205 and OE2-Glu222. Dashed blue lines connect atoms within H-bond distance, distances between oxygen atoms (in ˚ A) are written next to the lines (red for an unfavorable angle for a H-bond).

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Figure 14: Dynamics of water insertion between Ser205 and Glu222. Top: Ser205 χ1 dihedral angle, and bottom: length of the 205–222 PW, as a function of time. Length of 1 implies water insertion, while no data means no PW. Data for bottom plot were smoothed similarly to Figure 5. Inset: magnification of main the plot for times between 2 and 8 ns.

Table 4: 205–222 PW length for different Thr203 rotameric states correlates with the PWM exchange rates Thr203 state PW length PWM exchange/ns g+ 1.16 1.94 g0.99 0.59 t 0.60 0.58

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3.3.5

The water pool

The water molecule that gets inserted between Ser205 and Glu222 is one of the five crystal water molecules occupying a cavity under the chromophore plane (that includes residues Glu222, Ser72, and Gln69). This is one of two cavities inside the GFP β-barrel, which host a cluster of water molecules (the other cavity is located above the chromophore plane and was suggested 105 , together with neighboring protein atoms, to play a role in chromophore biosynthesis). We call these clusters “water pool 1” (WP1) and “water pool 2” (WP2), respectively. The five WP1 water molecules engage in a H-Bonding network. Figure 15 presents a scheme of this network before (4517 ps) and after (4679 ps) the first water insertion event. The high connectivity of WP1 water molecules can also be deduced from Table 5, which shows the number of H-bonds per water molecule for the different locations with respect to the protein surface. WP1 water molecules have a higher number of H-bonds to other water molecules (2.0 vs. 0.78 for other internal water molecules) and lower number of H-bonds to protein atoms (1.0 vs. 1.93 for the other internal water molecule). These values are closer to those calculated for surface water, although WP1 is located in the midst of the GFP barrel. Inspection of the trajectory reveals that the five water molecules are constantly exchanging places and H-Bonding partners. At one point, Wat2104 climbs up and inserts between OG-Ser205 and OE2-Glu222, breaking the direct H-bond between them and hence elongating the PW. The other four water molecules rearrange and Wat2106 takes the place of Wat2104 (see Movie S7 in the SI). During the process an additional water molecule H-bonds to Wat2070, compensating for the loss of a water molecule from WP1. Note, however, that cavity water molecules do not exchange as readily with the bulk as the PWM. The five cavity water molecules from the minimized X-ray structure Wat2070, Wat2104, Wat2106, Wat2109, and Wat2308 reside there until 87, 68, 12, 101, and 52 ns from the beginning of the simulation, respectively (compared to an average PWM time between exchange events of only 3.8 ns at 300 K). 70 Out of the 220 water molecules participating in the 205–222 PW during the trajectory, 93 originate from WP1 (the rest originate from the bulk through the PWM exchange pathway). 70 During the simulation, all five original crystal water molecules from WP1 climb up and participate in the 205–222 PW. These, in turn, are replaced by bulk water molecules entering from the barrel bottom, some of which participate in the 205–222 PW later on. The high mobility of WP1 water molecules 73 is supported by their high 35



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X-ray B-factors and Mean Squared Displacement (MSD) values during the MD trajectory, as discussed in Section S2.4 in the SI (see also Table S8 there). An additional indication of high mobility is that, unlike all other internal water molecules in the GFP crystal, two out of the five WP1 water molecules have double occupancy. The above observations can be connected with possible scenarios for PT within GFP. Water exchanging through the “hole in the barrel” may trace a possible exit pathway for the proton out of the GFP barrel. Likewise, the flow of water molecules from the barrel bottom via WP1 to Glu222 may trace a proton entry pathway. The higher coordinating number of water molecules in WP1 (and their lability) could be suitable for accommodating a hydrated proton, a central ansatz in our revised model of proton migration in GFP (below).

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Figure 15: Schematic drawing of the PW network from the chromophore to Ser72 and Gln69 before (left) and after (right) water insertion between Ser205 and Glu222. Representation of residues is simplified, showing only the functional group. Water molecules are presented with their residue numbers only (according to the PDB file notations) and colored for clarity. Protein atoms are presented with their name and residue number as subscript. H-bonds are shown in red dashed lines. Atom positions, bond distances, and angles were adjusted for clarity of representation. Note that Water7933 does not exist in the PDB file. It was added during the solvation stage, and is thus an example of a bulk water molecule that has entered WP1 during the simulation.

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Table 5: Number of H-bondsa for water molecules in the minimized 2WUR X-ray structure Type of water molecule WP1b Other internalc Surfaced Bulke

Number of H-bonds with: water molecules protein atoms 2.0 1.0 0.79 1.93 2.12 1.25 3.78 0

a

Calculated as an average over all water molecules from the group. b Five crystal water molecules (Wat2070, Wat2104, Wat2106, Wat2109, and Wat2308) occupying the cavity between Glu222 and Ser72. c Crystal water molecules located inside the GFP barrel (identified by visual inspection), excluding those of the previous group (total of 14 water molecules). d Water molecules not belonging to “WP1” or “other internal” and are H-bonded to at least one protein atom. e Water molecules which are H-bonded only to other water molecules.

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3.3.6

Water entry and water wire along the barrel axis

Our study of PWs near the chromophore revealed higher participation of water molecules than in the X-ray structures. Specifically, we found water insertion between strands 7 and 10 to form an exiting WW from OH-Cro66 to the bulk, and the insertion of an extra water molecule between Ser205 and Glu222. In addition, we witnessed that water transfer from WP1 to the 205–222 PW triggers entry of additional water molecules from the bottom of the GFP barrel along the barrel axis. It is therefore possible that the GFP crystals (as used for the X-ray measurements at low temperatures) are dehydrated as compared with MD at room temperature. Because the starting point for our simulations is the X-ray structure (specifically, the high-resolution PDB file 2WUR), we follow with time the number of water molecules around amino acid residues that are situated near the barrel axis. Figure 16 presents the number of water molecules neighboring (within ˚ 5 A) residues 68-72 from the central helix, as an estimate for the number of water molecules inside the lower part of the GFP barrel. The figure shows that this number increases from an initial value of ∼ 10 to a plateau value of ∼ 20, reached approximately 70 ns after the beginning of the simulation. The main contribution to the rise in the number of water molecules is from Cys70 and Phe71, which are closer to the barrel rim. It is therefore possible that some of this rise is due to their sidechains moving and getting exposed to bulk water. More representative, perhaps, is Ser72, around which we see the addition of about 5 water molecules. However, all residues show the increase from the initial value, suggesting that in its native state, the GFP barrel is uniformly filled with water molecules and the five crystal water molecules “survived” the crystallization process due to their more internal location. Perhaps in correlation with the increase in water content is the increase in backbone atoms RMSD with time, shown in Figure S4 of the SI. Either backbone movement allows water entry or, vice versa, internal waters “lubricate” the protein resulting in larger backbone RMSD. Commensurate with the above, we find a complete WW leading from Glu222 to the barrel bottom, exiting to the bulk. As seen in Figure 17, the WW emerging from OE2-Glu222 is composed of 6–7 water molecules until reaching the bottom exit, near Asp82. A random walk search for all PWs emerging from OE2-Glu222 was conducted. There, direct Glu222–bulk WW (pathways in which OE2-Glu222 serves as an exit point) exists as 1.5 % of all exit paths. The probability of such direct WWs is relatively low, this is because a random walk of 6–7 steps has a high chance of encountering a 39



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non-water atom along the way, thus terminating the all-water wire. This is shown in Figure 17 in which the WW passes near Asp82, Cys70, and Ser72 and forms H-bonds with them. Loosening the condition for an all-water PW, we find that general PWs from Glu222 to the bulk through a bottom exit constitute 29 % of all exit paths (see SI Table S9 for a list of protein atoms at the barrel bottom). Inspecting the most common protein atoms that serve as bulk exits (O-Cys70, OD1/2-Asp82, NZ-Lys79, NE2-His199, NZ-Lys85, and OH-Tyr74, termed generally as Pbottom ), we find that the most common paths leading to them are WWs of the form: OE2-Glu222 → W1 → W2 . . . Wn → Pbottom , where n is between 5 and 9. In cases where a protein atom is part of the wire, it is always a Pbottom atom. Thus, the barrel internal space hosts a column of water molecules that can serve as a WW. Neighboring protein atoms stabilize the WW and can serve as temporary resting spots.

˚ distance from Figure 16: Number of different water molecules within 5 A residues 68-72 on the internal α-helix. For water neighbors counting, all the atoms in the residue were included. For clarity of representation, data were smoothed in a way that the value at each time is an average of the data points within 10 ps preceding and following it.

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Figure 17: Snapshot taken from the simulation at 86.9 ns depicting a WW between OE2-Glu222 and the “bottom” of the protein, going out to the bulk.

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4

Summary

In this work, a > 300 ns MD simulation was used to explore PW dynamics inside wt-GFP, find out how these differ from the X-ray PWs, and hopefully shed light on the PT mechanism in GFP that could reconcile the conflicting evidence discussed above. The results shown here are limited to a protonated chromophore (in its ground electronic state) with a deprotonated Glu222, which is the state generally believed to characterize the X-ray structures. Indeed, we find several PWs and WWs not seen in wt-GFP X-ray structures. First, the “canonical” 66–222 PW is extended by an additional water molecule, W1 , inserted between Ser205 and Glu222 OH-Cro66 → PWM → OG-Ser205 → W1 → OE2-Glu222 , see Figure 13. The insertion occurs when Ser205 is in its t rotameric state (Figure 6C). To make room for W1 , Ser205 then rotates to its g- state (Figure 14). W1 arrives, with nearly equal probabilities, either from the bulk (through the “hole in the barrel” next to OH-Cro66) or from a cluster of 5 water molecules (WP1) located just below Glu222 (Figure 15). Alternate 66–222 pathways that bypass Ser205 altogether are seen in the “abnormal” Thr203 g+ state, which is normally not observed in GFP X-ray structures or MD simulations. Here it occurs only, perhaps, because our simulation is very long (g+ is seen after about 180 ns). Instead of Ser205, we then find on the 66–222 PW either one or two water molecules, or OG1-Thr203. The most striking observation involves two WWs in locations where, based on X-ray structures, PWs were previously suggested. 59 Thus dynamics does not obliterate these wires, it enhances them by providing pathways constructed exclusively from water molecules, which are expected to facilitate PT. One WW, constructed of 3 water molecules, connects OH-Cro66 with the bulk through the PWM and the “hole in the barrel” located between β-strands 7 and 10 (Figure 9). The formation of this exiting WW (in competition with the 66-222 wire) becomes more probable in the Thr203 t state, in which the bulky and hydrophobic threonine methyl group is closest to Ser205. This interferes with the Ser205 rotational freedom and with the PW through it to Glu222. The second WW, replacing the PW leading from Glu5 (at the barrel’s bottom) to Glu222, traverses the barrel’s axis connecting its bottom with WP1 (Figure 17). Why was this “axial WW” never seen in wt-GFP X-ray structures then? We have followed in time the number of internalized water molecules in the lower half of the barrel, finding that during the first 70 ns of the simulation their number nearly doubles (Figure 16). This suggests 42


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that this region of the protein, at least, was partly dehydrated in the protein crystal. The reason for this might reside with the solution from which the crystal has precipitated. For 2WUR it is 40 % ethanol + 10 % dioxane, which exerts an osmotic pressure that could partly dehydrate the protein interior (and enhance the X-ray resolution). Indeed, 7 ethanol (and one isopropanol) molecules are seen in the 2WUR PDB structure on the surface of the protein. Thus the PWs observed in the X-ray structures are rough sketches of the wires that exist in the fully hydrated protein. Interestingly, both WWs were observed in the X-ray structures of GFP-like fluorescent proteins (such as zRFP574, TurboGFP, KillerRed, asFP595, and the kindling FP). 64–68 Conserved residues in GFP-like proteins are considered vital to the (ancestral) protein function. 82 Our results suggest that these two WWs are themselves conserved entities, a corollary that would be interesting to check in future work. Since WWs are expected to facilitate the transport of either electrons or protons, the “original” function of the ancestral GFP may have been, as argued by Zimmer and coworkers, 82 electron or proton transport. The observation that a tyrosine residue (at location 66 in wt-GFP) is universally conserved was used to argue 82 that the electron transport role preceded that of PT, perhaps because phenolate is known to have a low ionization energy in water. Yet a tyrosine is also pivotal for creating a chromophore that can undergo ESPT, 40 because it is the only naturally occurring amino acid with an aryl-OH sidechain. Hence, the discovery of the exiting and axial WWs in wt-GFP raises again the question whether long-range PT takes place in this protein and how. 59 We conclude with a detailed discussion of this possibility.

5 5.1

Discussion The dilemma

PWs are considered the preferred mechanism by which protons move inside proteins. Based on the X-ray data, a model for PT within GFP was proposed, 44 in which the photodissociated proton travels along the “canonical” pathway, OH-Cro66→PWM→OG-Ser205→OE2-Glu222, to the anionic glutamate and resides there for the duration of the excited-state lifetime of the chromophore. According to early time resolved fluorescence measurements, this occurs in 10 ps. 39 Glu222 protonation paralleling OH-Cro66 deprotonation was indeed seen in time-resolved IR measurements, 47–50 though slower than predicted by model quantum PT simulations. 54,55 This simple scenario could not explain time resolved fluorescence from the GFP A-state, which 43



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showed a power-law tail extending to the ns regime, with t−1/2 asymptotics below 230 K, switching to t−3/2 at higher temperatures. 71,75 This behavior could follow from a 1D model for proton diffusion along a PW extending down beyond Glu222 (to the bottom of the barrel) and beyond OH-Cro66 (directly to the bulk). 59 Yet such a scenario conflicts with the high proton affinity of an anionic glutamate, which should arrest any further proton migration. 78 We now suggest a revised model for long-range PT in wt-GFP that is consistent with all the above data.

5.2

Is Glu222 anionic?

In the A-state GFP (neutral chromophore) Glu222 was always assumed to be anionic (glutamate). 44 This could not be verified by earlier X-ray studies, which had insufficient resolution for observing H atoms and/or resolving small differences in bond lengths. This issue can be revisited using the ultra-high wt-GFP X-ray structure (PDB file 2WUR, 0.9 ˚ A resolution). 72 Figure 18 shows a portion of the electron density maps near the chromophore. 72 The blue contours are the model without hydrogens, and the green contours are the difference between the observed and computed electron density, which could be either the (missing) hydrogens, or noise. First we note a small electronic density on the OE2-Glu222 atom (green) that might represent partial protonation. Then, on OG-Ser205 the OH hydrogen is very clear, and it points toward the PWM (Wat285) and not toward OE2-Glu222, as it would if Glu222 were anionic. Indeed, in our simulations (with an anionic Glu222), the Ser205 OH group rotates, already during the optimization stage, toward OE2-Glu222 (Figure S3A in the SI). The PWM, in turn, donates its H-bonds to Asn146 and Tyr66 (and not to Ser205). Thus, this PW has the opposite orientation than usually postulated. Next, consider the C–O bond lengths of Glu222. Ahmed et al. 91 have shown that if the X-ray resolution is better than 1.2 ˚ A, and the carboxylic oxygen atoms have an occupancy of 1 and B-factors < 10 ˚ A2 , glutamate and aspartate protonation states can be determined from the two C–O bond lengths. If they are equal (1.256 ˚ A) the Glu/Asp amino acid is anionic; If the two distances differ (one is a single bond, ca. 1.31 ˚ A, the other a double ˚ bond, ca. 1.21 A) these residues are protonated. Figure 19 shows the CD–OE1 and CD–OE2 distances for all 15 glutamate residues in the 2WUR structure. Excepting Glu222, all are surface residues with their sidechains protruding to solution, and they have occupancy < 1 or B-factors > 10 ˚ A2 . Therefore, these can deviate from bond-equality even when deprotonated. The largest deviation from bond equality is for Glu132, 44


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Figure 18: Hydrogen omit electron density maps of the chromophore region from Figure 4B of ref 72. Contour levels are 2.0 σ for both 2Fo − Fc (blue) and Fo −Fc maps. Hydrogen atoms (small gray spheres) were included in the final model for all C-H groups and for O-H and N-H groups with pKa > 7.0. with B > 30 ˚ A2 for its OE atoms. Glu222 is the only buried glutamate, and has small B-factors (7.7 and 6.0 ˚ A2 for its two OE atoms). Therefore its CD–OE distances should be accurately determined by the X-ray data. The difference between these two CD–OE bond lengths is 0.8 ˚ A, nearly as large as expected from a protonated carboxylic acid (1.0 ˚ A). The longest of the two is the CD–OE2 bond, and it is indeed the one pointing toward OG-Ser205. Thus Glu222 is not anionic, in agreement with the density maps in Figure 18. We conclude that at the low temperatures of the X-ray crystal (T = 100 K for 2WUR) Glu222 is protonated, but it gets deprotonated at room temperature, at which all IR measurements were performed thus far. Thermodynamically, the dissociation reaction is expected to exhibit positive enthalpy change (∆H > 0), because a chemical bond is cleaved, and positive entropy change (∆S > 0), because the dissociated proton can delocalize over a cluster of water molecules. Hence the free energy change in the reaction, ∆G = ∆H − T ∆S, becomes more negative as T increases, promoting dissociation. The proton that binds to Glu222 as the protein is cooled to 100 K

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Figure 19: The two carboxylic C–O distances (star and diamond symbols) for the 15 glutamate residues in the wt-GFP structure from PDB file 2WUR (0.90 ˚ A resolution). OEX represents atoms OE1 and OE2 of glutamate residues. Glu213 and 222 (black symbols) are the only residues with single occupancy and B-factors < 10 ˚ A2 , which are the required preconditions for determining the protonation state from such data. 91 B-factors, taken from the 2WUR X-ray structure, represent the average value for the OE1 and OE2 atoms. cannot arrive from far away. We suggest that it is hosted within the WP1 water cluster. Consequently, GFP proton relay involves two protons.

5.3

The two-proton scenario

With the realization of an extra proton in WP1, the sequence of events following photoexcitation may be envisioned as follows (Figure 20): a) ESPT is based on an intramolecular charge transfer (ICT) effect, where electronic charge in the proximal (here, Tyr66) ring moves toward the distal (imidazolidinone) ring. 106 Quantum calculations show that in the GFP chromophore, the electronic charge moves predominantly to the bridging 46


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carbon atom (CB2, see Figure S1 in the SI), rather than all the way to the imidazolidinone ring. 107,108 More specifically, the charge of the bridging atoms of the anionic chromophore goes from −0.04 in S0 to −1.28 in the S1 electronic state. 108 The more positive OH-Tyr66, and more negative CB2, now promote the motion of two protons, rather than a single proton as previously assumed. b) The reduced electron density on OH-Tyr66 triggers its deprotonation (PT1). This is the ESPT reaction observed by the time-resolved fluorescence measurements. c) The enhanced negative charge on CB2 promotes Glu222 protonation (PT2) by the proton hosted in WP1. This is the reaction observed by the time-resolved IR measurements. The average distance between CB2 and the OE moieties of Glu222 is about 5 ˚ A, sufficiently close to exert a strong effect. d) The dissociated proton leaves the GFP barrel via the exiting WW (Figure 9) when the temperature is sufficiently high. At lower temperatures this proton diffuses along the internal WW (Figure 17), giving rise to the power-law in the OH-Tyr66 fluorescence. Note that Glu222 is now protonated, and thus cannot interfere with the internal proton mobility. d) Back in the ground-state, Glu222 transfers its proton to − O-Tyr66, while a new proton is recruited by the “proton antenna” and channeled via the axial WW to WP1. This completes the GFP PT cycle. The elegance of this mechanism is that a single photon moves two protons along the route WP1→Glu222→ Tyr66→bulk: One proton moves from WP1 to Glu222 as the other goes from Tyr66 to the bulk. This tentative mechanism, which is consistent with all known data, should be investigated in future work.

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Figure 20: Suggested mechanism for light-induced double-PT in wt-GFP, when the chromophore (Tyr66+CB2+imidazolidinone) is in its excitedstate. See text for detail.

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Acknowledgment. Work supported by THE ISRAEL SCIENCE FOUNDATION (grant number 766/12). The Fritz Haber Research Center is supported by the Minerva Gesellschaft f¨ ur die Forschung, M¨ unchen, FRG.

5.4

Supporting Information

Supporting figures (S1, GFP chromophore scheme; S2, flowchart for the random walk PW search algorithm; S3, superimposed structures of X-ray, minimized, and after 14 ns of MD simulation; S4, RMSD of protein and distances between strands 7-10; S5–S7, 205–222 PW length and S205/T203 dihedral angles for simulations for different PDB structures). Supporting tables (S1–S5, force field parameters for GFP chromophore; S6, Iavg for protein atoms H-bonded to WW atoms; S7, T203 dihedral states at S205 rotation and 205–222 PW water insertion; S8, B-factors for crystallographic water molecules; S9, list of atoms at the barrel “bottom”). Supporting movies (S1, Thr203 sidechain rotation and T203-E222 H-bond formation; S2, Glu222 tilting its sidechain away from Ser205; S3, Ser205 rotating toward Glu222; S4-S6, Rotational view of Figures 11A-C; S7, water insertion between Ser205 and Glu222). This information is available free of charge via the Internet at http://pubs.acs.org

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Figure 21: For Table of Contents Only

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