Supporting Information Proton Wire Dynamics in the Green ...vintage.fh.huji.ac.il/~agmon/Fullpaper/ct6b00939_si_001.pdf · Proton Wire Dynamics in GFP, supporting information S5 Table

Supporting Information

Proton Wire Dynamics in the Green Fluorescent

Protein

Ai Shinobu and Noam Agmon*

The Fritz Haber Research Center, Institute of Chemistry, The HebrewUniversity of Jerusalem, Jerusalem 91904, Israel

*E-mail: [email protected]

Contents

S1 Methods S3

S2 Results and analysis S10S2.1 Backbone mobility in MD simulations . . . . . . . . . . . . . S10S2.2 Random walk WW from OH-Cro66 path . . . . . . . . . . . . S12S2.3 205–222 PW water insertion, correlation with S205/T203 ro-

tations for multiple short MD trajectories . . . . . . . . . . . S13S2.4 Mobility of WP1 water molecules . . . . . . . . . . . . . . . . S18

S3 Supporting movies S20

List of Figures

S1 GFP chromophore . . . . . . . . . . . . . . . . . . . . . . . . S3S2 Random walk PW search algorithm flowchart . . . . . . . . . S9S3 Superimposed chromophore region, X-ray, minimized, and MDS10S4 RMSD and strand 7-10 distances . . . . . . . . . . . . . . . . S11S5 205–222 PW length, T203/S205 dihedrals, #1 . . . . . . . . . S14S6 205–222 PW length, T203/S205 dihedrals, #2 . . . . . . . . . S15S7 205–222 PW length, T203/S205 dihedrals, #3 . . . . . . . . . S16

S1

Proton Wire Dynamics in GFP, supporting information S2

List of Tables

S1 GFP chromophore atoms names and types . . . . . . . . . . . S4S2 Bonded interactions parameters, neutral chromophore . . . . S5S3 Angular interaction parameters, neutral chromophore . . . . S6S4 Dihedral interaction parameters, neutral chromophore . . . . S7S5 Improper dihedral interaction parameters, neutral chromophore S8S6 Properties of protein atoms H-bonding to WWs from OH-Cro66S12S7 Relation between 205-222 PW water insertion and S205 di-

hedral rotation . . . . . . . . . . . . . . . . . . . . . . . . . . S17S8 B-factors for crystallographic water molecules . . . . . . . . . S18S9 List of Pbottom atoms . . . . . . . . . . . . . . . . . . . . . . . S19


S1 Methods

Figure S1: GFP chromophore. Atom names are written in PDB format andcolored according to original residue: Ser65 (green), Tyr66 (blue), Gly67(red).

The following five tables list the force field parameters for the chro-mophore, adopted from Ref. 87 (in main text).


Table S1: List of the GFP chromophore atoms namesand types

Atom name inPDB formata

Atom type inCharmm format Atom charge, e

CB1 CT2 0.05HB11 HA 0.09HB12 HA 0.09OG2 OH1 -0.66HOG H 0.43N1 NH1 -0.47

HN11 H 0.31CA1 CT1 0.07HA1 HB 0.09C1 CPH2 0.76N2 NR2 -0.55N3 NR1 -0.64C2 CPH1 0.80O2 O -0.61

CA2 CPH1 0.24CB2 CE1 -0.1HB21 HA1 0.1CG CA 0CD1 CA -0.12HD1 HP 0.12CD2 CA -0.12HD2 HP 0.12CE1 CA -0.12HE1 HP 0.12CE2 CA -0.12HE2 HP 0.12CZ CA 0.11OH OH1 -0.54HO H 0.43CA3 CT2 -0.18HA31 HA 0.09HA32 HA 0.09

C3 C 0.51O3 O -0.51

a Following the IUPAC-IUB rules, as described inRef. 51.


Table S2: Force field parameters for bondedinteractionsa for the neutral chromophore

Atom1 type Atom2 type Kb, kcal/mol/A2 b0, A

CT1 CPH2 320 1.49NR1 CT2 352 1.45NR1 CPH2 400 1.39NR1 CPH1 400 1.41

CPH1 O 807 1.22NR2 CPH2 400 1.3

CPH1 CPH1 410 1.49NR2 CPH1 400 1.4CE1 CPH1 560 1.36CE1 HA1 360.5 1.1CA CE1 370 1.45CA CA 305 1.38HP CA 340 1.08

OH1 CA 334.3 1.41OH1 H 545 0.96

a Bond potential is of the form: V (bond) = Kb(b−b0)2,where b is the distance between atom1 and atom2 inCharmm format, Taken from Ref. 87.


Table S3: Force field parameters for angular interactionsa for the neutralchromophore

Atom1 type Atom2 type Atom3 type Kθ, kcal/mol/rad2 θ0, deg

CPH2 CT1 HB 50 109.5NH1 CT1 CPH2 50 107

CPH2 CT1 CT2 52 108CT1 CPH2 NR2 40 125NR2 CPH2 NR1 130 114

CPH2 NR2 CPH1 130 106CT1 CPH2 NR1 35 121.4

CPH2 NR1 CPH1 130 107.9CPH2 NR1 CT2 36 129NR2 CPH1 CPH1 130 108.3CE1 CPH1 NR2 45.8 129.5

CPH1 NR1 CT2 32 123.4NR1 CPH1 O 42 126NR1 CPH1 CPH1 130 103NR1 CT2 HA 48 108

C CT2 NR1 50 107CPH1 CPH1 O 38 132CE1 CPH1 CPH1 45.8 122

CPH1 CE1 CA 130 130CPH1 CE1 HA1 42 114CE1 CA CA 45.8 121HA1 CE1 CA 42 116OH1 CA CA 45.2 120

H OH1 CA 65 108CA CA CA 40 120HP CA CA 30 120

OH1 CA CA 45.2 120H OH1 CA 65 108

a Angular potential is of the form: V (angle) = Kθ(θ − θ0)2, where θ is

the atom1-atom2-atom3 angle. Atoms listed are in Charmm format.Taken from Ref. 87.


Table S4: Force field parameters for dihedral interactionsa for the neutralchromophore

Atom1type

Atom2type

Atom3type

Atom4type Kχ, kcal/mol n, multiplicity δ, deg

CA CA CE1 CPH1 1.4 2 180CA CE1 CPH1 CPH1 6.84 2 180

CPH1 CPH1 CE1 HA1 6.84 2 180C CT2 NR1 CPH1 0.067 3 180

CPH1 NR1 CT2 HA 0.067 3 180C CT2 NR1 CPH2 0.067 3 0

CPH2 NR1 CT2 HA 0.067 3 180CA CA CE1 HA1 1.4 2 180CT2 CT1 CPH2 NR1 0.1 3 0HB CT1 CPH2 NR1 0.1 3 0NH1 CT1 CPH2 NR1 0.1 3 0NR2 CPH1 CE1 CA 6.84 2 180NR2 CPH1 CE1 HA1 6.84 2 180CT2 CT1 CPH2 NR2 0.1 3 180HB CT1 CPH2 NR2 0.1 3 180NH1 CT1 CPH2 NR2 0.1 3 180

a Dihedral potential is of the form: V (dihedral) = Kχ(1 + cos(n(χ − δ))),where χ is the atom1-atom2-atom3-atom4 dihedral angle. Atoms listedare in Charmm format. Taken from Ref. 87.


Table S5: Force field parameters for improper dihedral interactionsa for the neutral chro-mophore

Atom1 type Atom2 type Atom3 type Atom4 type Kψ, kcal/mol/rad2 ψ0, deg

CPH2 CT1 NR1 NR2 0.5 0NR1 CPH1 CPH2 CT2 0.45 0

CPH1 NR1 CPH1 O 0.5 0CPH1 CPH1 CE1 NR2 220 0CE1 CPH1 CA HA1 30 0

a Improper potential is of the form: V (improper) = Kψ(ψ − ψ0)2, where ψ is the

improper angle around atom1. Atoms listed are in Charmm format. Taken fromRef. 87.


Figure S2: Flowchart describing the random walk PW search algorithm.“A(0)” denotes the starting atom of the search (in the current case,OH-Cro66 or OE1/2-Glu222) “T” and “F” written next to arrows denote“True” and “false”, respectively.


S2 Results and analysis

S2.1 Backbone mobility in MD simulations

Figure S3: Superposition of the chromophore region comparing (A) 2WURX-ray structure (in green) and the energy-minimized structure, (B) theenergy-minimized structure (in orange) and a snapshot from the MD simu-lation at 14 ns. Structures were fitted with respect to the backbone atomsof residues Cro66, Ser205, Thr203, Glu222, and His148. Distances betweenselected atoms (in A) are shown next to the dashed lines connecting them,according to the format (A) X-ray/minimized, (B) minimized/MD.


Figure S4: Top: RMSD, calculated for backbone atoms, compared to X-raycoordinates, for all residues in protein except the termini residues 3-9 and227-232 (black), and for residues 129-132. Bottom: Distances betweenresidues in opposing strands 7-10, residue pairs shown in legends. Distancewas measured between the backbone nitrogen atom of the first residue, andthe backbone oxygen atom of the second residue.


S2.2 Random walk WW from OH-Cro66 path

Table S6: Protein atomsa H-bonding to WWsfound with a random walk PW search fromOH-Cro66

Atom name Iavgb

layer inPWM

exchangecfraction ofappearanced

OG1-Thr203 1.01 1 0.02OG-Ser205 1.04 1 0.10O-Asn146 1.36 1 0.06N-Ser205 1.93 2 0.11O-Ser205 2.35 2 0.10N-Tyr145 2.48 2 0.03O-Tyr145 2.78 2 0.10N-Leu207 3.68 2 0.03

OG-Ser147 3.71 2 0.03N-Ser147 4.91 2 0.02

OE1-Gln204 5.25 3 0.02NE2-Gln-204 5.48 3 0.03OD1-Asn-144 5.69 3 0.02ND2-Asn-144 6.05 3 0.02

O-Leu-207 6.69 3 0.02

a Atoms shown here participate in > 2 % ofall H-bonds between protein atoms and WWwater molecules.

b Defined in the main text.c As defined in section 3.2 in Ref. 69, see Fig-

ure 8 in the Ref. 69.d Fraction of H-bonds with WW atoms for the

specific protein atom from the total numberof H-bonds created by WW atoms and pro-tein atoms.


S2.3 205–222 PWwater insertion, correlation with S205/T203rotations for multiple short MD trajectories

In the current trajectory only few Thr203 rotations occur, making it difficultcharacterize the phenomenon. In an attempt to link between water insertionand the rotameric states of Thr203 and Ser205 we inspect data from multiplesimulations performed in our previous work (a total of 17, 18 ns simulationsin five different temperatures 300, 310, 320, 330, and 340 K, Ref. 69). There,three different crystal structures (2WUR, 1EMB, and 1EME) were used asinitial structures and they differed in their Thr203 rotameric state. The2WUR structure (also used in the current work) and 1EMB have Thr203in the less common initial state of g- (in 1EMB Thr203 appears in doubleoccupancy but the more occupied g- state was used for simulation). The1EME structure had Thr203 in the t state. All three X-ray structures haveSer205 in the initial rotameric state of t.

Rotations of Thr203 and Ser205 occur in most simulations, althoughsimulations of the 1EME initial structure sample less of the Thr203 g- state(36 % of the time comparing to 59 % and 61 % for 1EMB and 2WUR, re-spectively). Water insertion in the 205–222 PW also occur in all but 4 ofthe individual simulations. Inspection of the rotameric states of Thr203and Ser205 at times of water insertion shows that all insertions occur whenThr203 is in the g- state. Regarding Ser205, most insertions (10 out of 13)occur in proximity (less than 50 ps apart) to its first rotation from state t.The rest of the insertion events occur in the t state of Ser205. Figures S5–S7and Table S7 present detailed water insertion data for all 17 simulations. In-spection of times of Ser205 rotation and water insertion with respect to eachother does not provide a clear answer to which of the events activate theother. However, it is clear that both processes are favored by Thr203 beingin the g- state. Most past MD works on GFP used initial structures withThr203 in the more common t state. It is not clear if they even sampleother Thr203 states. The current simulation, due to its unusual Thr203conformation, managed to detect the uncommon event of water insertion inthe 205–222 PW.


Figure S5: Panels showing 205–222 PW length (top figure in each panel) andT203/S205 dihedral angles (middle and bottom in each panel, respectively)for 18 ns MD simulations performed in Ref. 69 from main text. Initial PDBstructure and temperature of the simulation are written in the headline ofeach panel. The “*” sign in the headline denotes additional simulation forthe same initial PDB structure at the same temperature.


Figure S6: See legends of figure S5.


Figure S7: See legends of figure S5.


Table S7: between 205-222 PW water insertion and S205 dihedralrotation events for different MD simulationsa .

Initialstructure T , Kb ∆t(insert′ − rot′)c, ps S205 state in tinsert2WUR 300 −2333 t2WUR 300d 201 t2WUR 310 no event no event2WUR 310d −362.5 t2WUR 320 1 g-2WUR 330 −393 t2WUR 340 −1895 g-1EMB 300 -486 t1EMB 310 1.5 g-1EMB 320 1 t1EMB 330 -715.5 t1EMB 340 −22.5 t1EME 300 no event no event1EME 310 0 g-1EME 320 63 t1EME 330 0.5 g-1EME 340 −7 t

a Simulations performed in Ref. 69 from the main text.b Temperature of simulation.c ∆t between time of first water insertion in the 205–222 PW

and closest Ser205 rotation.d Additional simulation at the same temperature.


S2.4 Mobility of WP1 water molecules

X-ray data and root mean square deviation (RMSD) measurements duringthe simulation show that WP1 water molecules are more mobile than the restof the internal water molecules. Table S9 shows the average B-factor (B)of the crystallographic water molecules from the 2WUR X-ray structure.This measure of the atom mobility is related to the RMSD of the atom

by: RMSDX-ray =√

B8π2 . The table shows that the five WP1 cavity water

molecules have a higher B-factor in comparison to the other internal watermolecules (8.69 A2 vs. 5.77 A2). Moreover, two of the water molecules fromthe pool, Wat2109 and Wat2308, have two possible locations with occupancyvalues of 0.57 and 0.43. Their increased mobility during the X-ray structureacquisition might be an indicator of greater mobility of them during thesimulation, resulting in an increased rearrangement of their H-bonds. TheMD RMSD (RMSDMD) values of the internal water molecules were measuredfrom the beginning of the simulation until the time they left to the bulk.For the 5 WP1 water molecule the average RMSDMD was 0.67 A whereasfor the other internal water molecules it was 0.61 A, showing that the WP1water molecules are indeed more mobile during the simulation. The greatermobility of these water molecules is manifested in a more frequent H-bondexchange, which allows for an occasional water molecule to jump to theplane above Glu222, and be inserted between Ser205 and Glu222.

Table S8: B-factors for crystallographicwater moleculesa

Type of water moleculeb B-factor, A2

WP1 8.69Other internal 5.77

Surface 26.72

a Calculated as an average over all wa-ter molecules from the group. Valuestaken from the 2WUR X-ray struc-ture.

b As defined in Table 5 in the main text.


Table S9: List of Pbottoma atoms

N-LYS-3 OD1-ASP-76 N-THR-186O-LYS-3 OD2-ASP-76 OG1-THR-186O-GLY-4 NE2-HSD-77 O-LEU-194N-GLU-5 O-HSD-77 O-LEU-195

OE1-GLU-5 N-MET-78 O-PRO-196OE2-GLU-5 O-MET-78 N-ASP-197

O-GLU-5 SD-MET-78 O-ASP-197OE1-GLU-6 NZ-LYS-79 OD1-ASP-197OE2-GLU-6 O-LYS-79 OD2-ASP-197

N-LEU-7 N-ARG-80 ND2-ASN-198N-PHE-8 NE-ARG-80 O-ASN-198O-PHE-8 NH1-ARG-80 OD1-ASN-198O-THR-9 NH2-ARG-80 ND1-HSD-199O-GLY-10 O-ARG-80 NE2-HSD-199N-ASP-36 ND1-HSD-81 N-HSD-199

OD1-ASP-36 NE2-HSD-81 O-HSD-199OD2-ASP-36 O-HSD-81 OE1-GLU-222

O-ALA-37 N-ASP-82 OE2-GLU-222N-THR-38 O-ASP-82 N-ALA-226

OG1-THR-38 OD1-ASP-82 O-ALA-226O-THR-38 OD2-ASP-82 N-GLY-228

OH-TYR-39 N-PHE-83 O-GLY-228O-TYR-39 N-PHE-84 N-ILE-229N-CYS-70 N-LYS-85 O-ILE-229O-CYS-70 NZ-LYS-85 N-THR-230

SG-CYS-70 O-LYS-85 OG1-THR-230NE-ARG-73 N-SER-86 O-THR-230

NH1-ARG-73 OG-SER-86 ND1-HSD-231NH2-ARG-73 O-MET-88 NE2-HSD-231

N-TYR-74 O-MET-153 N-HSD-231OH-TYR-74 OD1-ASP-155 O-HSD-231O-TYR-74 OD2-ASP-155 N-GLY-232O-PRO-75 NE2-GLN-157 OT1-GLY-232N-ASP-76 O-ASN-159 OT2-GLY-232O-ASP-76 O-GLY-160

a Defined in section 3.3.6 in the main text. Bot-tom atoms were identified by manual inspectionof the X-ray structure.


S3 Supporting movies

S1: Rotation of Thr203 sidechain to the g+ dihedral state, allowing theformation of a H-bond between (OG1-T203)-(OE2-E222). Thr203 is shownin the bottom left, Glu222 in the bottom right. Chromophore is on top.Frames taken from the MD simulation between 208.9995 ns and 209.0995 nsS2: Rotation of Glu222 (bottom right) sidechain. Frames taken from theMD simulation between 112.056 ns and 112.614 ns.S3: Rotation of Ser205 (bottom left) to its t dihedral state and the creationof a H-bond to Glu222 (bottom right), chromophore rests on top. Framestaken from the MD simulation between 121.7895 ns and 122.2395 ns.S4, S5, and S6: Rotational view of Figures 11A, B, and C, respectively, fromthe main text, presenting the chromophore, Ser205, Thr203, and Glu222.S7: Water 104 inserts between Ser205 and Glu222, breaking the directH-bond between them and elongating the PW. Frames taken from the MDsimulation between 4.517 ns and 4.679 ns.

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Supporting Information Proton Wire Dynamics in the Green ...vintage.fh.huji.ac.il/~agmon/Fullpaper/ct6b00939_si_001.pdf · Proton Wire Dynamics in GFP, supporting information S5 Table