docking-guided identification of protein hosts for gfp ... · docking-guided identification of...

1

Supplementary information

Docking-guided identification of protein hosts for GFP chromophore-like

ligands

Natalia V. Povarovaa, Nina G. Bozhanovaa, Karen S. Sarkisyana, Roman Gritcenkoa, Mikhail

S. Baranova,b, Ilia V. Yampolskya,b, Konstantin A. Lukyanova, Alexander S. Mishina

aShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997

Moscow, Russia bPirogov Russian National Research Medical University, Ostrovitianov 1, Moscow 117997,

Russia

Supplementary file. Zip-archive with optimized geometries of Kaede chromophores.

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry C.This journal is © The Royal Society of Chemistry 2016

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Supplementary data and figures.

Table S1. Top-50 of candidate proteins assessed by molecular docking with GFP

chromophore.

Kaede docking

GFP docking

PDB ID Score PDB ID Score

1DOS -12.5 2GFX -9.75

1GVH -11 2QRY -9.6

1PVS -10.8 1DOS -9.6

3FBR -10.8 3HNZ -9.55

1VB6 -10.7 1XDQ -9.45

3ASV -10.4 2GFY -9.45

1TJ1 -10.2 3DNT -9.2

1V9Z -10.2 3FBR -9.15

3NR0 -10.2 3HO2 -9.15

1TLZ -10.2 3G11 -9.1

1TLY -10.2 1GVH -9.05

1TLW -10.2 1B3N -9.05

3IP0 -10.2 3I8P -9.05

2R46 -10.2 2GFV -9

1TJ2 -10.2 3EPS -9

3OW7 -10.1 1HO5 -9

1RP7 -10.1 1KFY -9

2FQ1 -10.1 2CGL -9

3DNT -10.1 3HZI -8.95

1I8T -10.1 1XDY -8.95

2FZM -10.1 2FDK -8.95

1FFT -10.1 2PUA -8.9

2ANB -10.1 3HO9 -8.9

2UDP -10 3MR8 -8.9

2QCU -10 3ASV -8.85

1TIW -10 2PUE -8.85

2FZN -10 2CGJ -8.85

1TJ0 -10 1IL2 -8.8

1MPG -10 1TJ2 -8.75

2CGL -9.9 2WDR -8.75

2R45 -9.9 1MWJ -8.75

1L8A -9.9 1FDI -8.7

1MWJ -9.9 3O7Q -8.7

1TLC -9.9 1PNS -8.7

1W7K -9.9 1K0G -8.7

3DMQ -9.9 3NQ8 -8.7

3Q2D -9.8 2WS3 -8.7

3D4V -9.8 2R4E -8.65

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3SEX -9.8 2ANB -8.65

3ITG -9.8 1K87 -8.65

2OWO -9.8 1NEN -8.65

1NAI -9.8 2WDV -8.6

3CW7 -9.8 2FZN -8.6

2R4J -9.8 2HGP -8.6

1LQA -9.8 1HPU -8.6

1W78 -9.8 2Q29 -8.6

2G28 -9.8 2QOW -8.6

2VET -9.8 1TLC -8.6

1K87 -9.8 3OAS -8.6

3CVS -9.8 1SPA -8.6

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Fig. S1 Spectral changes upon binding of the chromophore to proteins (1PVS, 3HO2, BSA).

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Table S2. Spectral properties of the tested chromophores in water solution (PBS pH 7.4).

Compound

λexc (nm) free/

+1PVS/ +3HO2

λem (nm) free/

+1PVS/ +3HO2

ε (M*cm

-1)

Solubility, µM

𝜙 (%)

λAbs (nm)

λAbs anion (nm)

PBS PBS +1% EtOH

PBS +5% EtOH

A05 478 475 480

568 563 565

31000 29.6 ND ND 0.40 468 525

A12 520 503 520

608 601 599

39500 1.1 10.5 13.8 0.05 481 ND*

A12H 480 481 485

572 577 576

35000 5.9 29.9 ND 0.03 465 ND*

A24 440 438 433

535 512 506

34500 8.4 13.5 31.0 0.15 436 511

*- multiple anion forms are possible.

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Fig. S2 Comparison of docking poses of the chromophores within the protein host 3HO2.

Chromophores A12 (A), A12H (B), A5 (C), A24 (D). Top-scoring mode is thicker, top 50

chromophore binding modes are shown as overlaid magenta sticks. Grey surface

corresponds to the binding pocket.

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Table S3 Residues with maximum impact on the ligand binding according to Rosetta ΔΔG

score. HI - hydrophobic interaction, 𝜋-𝜋 - stacking, ⋅⋅⋅H - hydrogen bond.

3HO2 1PVS

residue role ΔΔG residue role ΔΔG

A5 F399 𝜋-𝜋 -2.30 Y239 𝜋-𝜋 -1.48

F397 𝜋-𝜋 -1.42 T219 ⋅⋅⋅H -1.05

P271 ⋅⋅⋅H -1.08 W218 𝜋-𝜋 -2.93

L125 HI -1.64

A12 F399 𝜋-𝜋 -2.15 Y239 𝜋-𝜋 -2.10

D264 ⋅⋅⋅H -1.12 W218 𝜋-𝜋 -2.40

L125 HI -1.14

A12H F399 𝜋-𝜋 -2.19 Y239 -1.57

D264 ⋅⋅⋅H -1.12 W218 𝜋-𝜋 -2.61

L125 HI -1.10

A24 F399 𝜋-𝜋 -2.33 Y239 -1.88

T304 ⋅⋅⋅H -1.55 T219 ⋅⋅⋅H -1.11

P271 ⋅⋅⋅H -1.18 W218 𝜋-𝜋 -2.46

L125 HI -1.41

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Fig. S3 TDDFT studies on anionic and neutral species of A12H chromophore. (A)

experimental absorption spectrum; (B) Computed transitions for possible species of a12h in

solution, obtained at ZORA–PBE0/def2-TZVP (COSMO: H2O) level of theory, dashed lines

shows correspondence between theoretically predicted S0→S1 transitions and experimental

spectrum; (C) proposed equilibrium for a12h; (D) molecular orbitals (HOMO and LUMO) of

a12h_1_anion_2 and their contribution to S0→S1 transition.

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Fig. S4 TDDFT studies on anionic and neutral species of A12 chromophore.

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Supplementary Methods.

Table S4. List of primers used for self-assembly cloning

13.1 1DOS external forward

GGCCCGACGATACAGGACAAGAGACATGTCTAAGATTTT

TGATTTCGTAAAACCTGGCG

13.2 1DOS external reverse

AGACCCGCAGAGCGGGCCTTGAGATAAGCAGAAAGGAA

TATCTTACAGAACG

13.3 1DOS internal FL

CCGAGCTCGAGATCTATGTCTAAGATTTTTGATTTCGTAA

AACCTGGCGTAATCACTGGTGATGACGTACAG

13.4 1DOS internal FS

ATGTCTAAGATTTTTGATTTCGTAAAACCTGGCGTAATCA

CTGGTGATGACGTACAG

13.5 1DOS internal RL

CAGCCAAGCTTTTACAGAACGTCGATCGCGTTCAGTTCC

TGGAATGCTTTCTCCAGACGAGCG

13.6 1DOS internal RS

TTACAGAACGTCGATCGCGTTCAGTTCCTGGAATGCTTT

CTCCAGACGAGCG

13.39 2QRY FL

AGCTCGAGATCTATGTCTGCCCCTGCTGTTGCTGTGACA

GCGCCCG

13.40 2QRY FS ATGTCTGCCCCTGCTGTTGCTGTGACAGCGCCCG

13.41 2QRY RL

ACAGCCAAGCTTTTAACGGCTGACGGCGCGTTGCCATTC

GC

13.42 2QRY RS TTAACGGCTGACGGCGCGTTGCCATTCGC

13.38 1PVS RS TCATGCTTCGTCTGGTTGCCAGCCTTCCGTATACCAG

13.37 1PVS RL

ACAGCCAAGCTTTCATGCTTCGTCTGGTTGCCAGCCTTC

CGTATACCAG

13.36 1PVS FS

ATGTATACCCTGAACTGGCAGCCGCCGTATGACTGGTC

GTGG

13.35 1PVS FL

AGCTCGAGATCTATGTATACCCTGAACTGGCAGCCGCC

GTATGACTGGTCGTGG

13.13 3HO2 external forward

GTCCCACTAGAATCATTTTTTCCCTCCCTGGAGGACAAA

CGTGTCTAAGCGTCGTG

13.14 3HO2 external reverse

GGCCCGCAAGCGGACCTTTTATAAGGGTGGAAAATGAC

AACTTAGATCTTTTTAAAG

13.15 3HO2 internal FL

CCGAGCTCGAGATCTGTGTCTAAGCGTCGTGTAGTTGTG

ACCGGACTGGGCATGTTGTC

13.16 3HO2 internal FS

GTGTCTAAGCGTCGTGTAGTTGTGACCGGACTGGGCAT

GTTGTC

13.17 3HO2 internal RL

CAGCCAAGCTTTTAGATCTTTTTAAAGATCAAAGAACCAT

TAGTGCCACCGAAGCCG

13.18 3HO2 internal RS

TTAGATCTTTTTAAAGATCAAAGAACCATTAGTGCCACCG

AAGCCG

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Materials

Commercially available reagents were used without additional purification. For column

chromatography, E. Merck Kieselgel 60 was used. NMR spectra were recorded on a 700-

MHz Bruker Avance III NMR at 293 K. Chemical shifts are reported relative to residue peaks

of DMSO-d6 (2.51 ppm for 1H and 39.5 ppm for 13C). Melting points were measured on a

SMP 30 apparatus. High-resolution mass spectra were obtained on a Thermo Scientific LTQ

Orbitrap.

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Geometry optimization

Geometry optimization of chromosphere models was performed in ORCA 3.0.3

software 1 in the framework of DFT theory. RKS B3LYP hybrid DFT functional has already

been shown sufficient for geometry optimization of related compounds 2–4. TurboMole

program system defined B3LYP version was used in the present studies. def2-SVP and ma-

Def2-SVP basis set were tested and no significant difference was obtained, so def2-SVP

basis set was chosen to perform geometry optimization in order to save a computational

time compared to its analog with diffuse functions5,6. Split-RI-J method in conjugation with

“chain of spheres” COSX approximation (RIJCOSX) was successfully applied in order to

speed up calculations 7. COSX grid was tightened up to GRIDX4 to prevent numerical noise

appearing issues. For RIJCOSX approximation corresponding auxiliary def2-SVP/J basis set

was used 8. Multigrid option was turned on 8, so for SCF iterations GRID4 was used and

gradients and final energies were obtained at FINALGRID5. Optimization was performed in

internal coordinates in vacuum with tightened TIGHTOPT criteria (Energy Change 1.0000e-

06 Eh, Max. Gradient 1.0000e-04 Eh/bohr, RMS Gradient 3.0000e-05 Eh/bohr, Max.

Displacement 1.0000e-03 bohr, RMS Displacement 6.0000e-04 bohr).

Time-dependent density functional theory calculations

TD–DFT studies were performed on geometries used for docking studies and

obtained at B3LYP/def2–SVP level of theory in gas phase. Tamm-Dancoff approximation as

well as RIJCOSX were successfully applied in order to speed up calculations. Only 10 first

excitations were computed, the size of the expansion space was set to 100. SCF and GRID

settings were kept the same as for geometry optimization, except otherwise noticed (ma–

def2–TZVP and aug–SVP calculations, where GRIDX5, GRID5, and FINALGRID6 grid

settings were used, and auxiliary basis set was decontracted, SCF convergence criteria

were tightened with VERYTIGHTSCF option). Basis set optimization using B3LYP hybrid–

GGA functional was performed. def2–SVP and def2–TZVP basis sets give almost no

differences for the first transition, addition of polarization functions (def2–TZVPP), led to no

observable changes (Fig S7,A). Relativistic effects were found to play minor role, only small

changes of computed excitations were observed below ~ 400 nm when ZORA model was

applied, for def2–TZVP basis set changes were smaller than for smaller def2–SVP (Fig

S7,B), this almost didn’t lead to increasing of computational time. Effect of diffuse functions

also was studied (Minimally augmented for def2-XVP and bigger augmented one for SVP),

similar to latter comparison, for bigger def2–TZVP basis set influence of diffuse functions is

smaller than for def2–SVP, but in both cases changes are minor, especially for the first

S0→S1 transitions, where the difference is no more than few nm (Fig S7, C), in the case of

ma–def2–TZVP basis set some issues, rising from nonsufficient grid size, were noticed (Fig

S7, D).

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Fig. S7 TDDFT basis set optimization on a12h_2_anion_2.

Basis set optimization revealed def2–TZVP to be sufficient for our studies. Further

DFT functional optimization showed, that transitions, obtained with hybrid-GGA functionals (Fig. S8,A), are red–shifted in comparison to range separated ones (Fig. S8,B), except BHANDHLYP (Fig. S8,C), which has quite big amount of HF exchange. The main disadvantage of range–separated functionals is their computational cost, because they can be used only in conjugation with RIJONX approximation, which is significantly slower than RIJCOSX. All methods predict transitions of anionic forms to be closer to the maximum in the experimental excitation spectrum, in comparison to neutral ones for a12h_2 set of molecules (Fig. S8,C), but to select exactly which one is very difficult, because of DFT functional choice dependence. So, PBE0 hybrid–GGA functional was used in present studies.

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Fig. S8 Comparison of different DFT functionals. (A) Screening of hybrid-GGA functionals

on a12_2_anion_2; (B) Screening of range separated functionals on a12_2_anion_2; (C)

Comparison of different functionals on a12h_2 set of molecules, anionic as well neutral

ones.

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Synthesis

((Z)-4-(4-hydroxybenzylidene)-2-methyl-1H-imidazol-5(4H)-ones (1)

((Z)-4-(4-hydroxybenzylidene)-2-methyl-1H-imidazol-5(4H)-ones (1) was synthesized as

reported previously 9 .

(Z)-4-(4-hydroxybenzylidene)-1-methyl-2-((E)-styryl)-1H-imidazol-5(4H)-ones (A) 10

To the solution of compound 1 (1 mmol) and corresponding aldehyde (1.2 mmol) in THF

(5 mL) anhydrous zinc chloride (30 mg, 0.22 mmol) was added. The mixture was refluxed for

1 h and the solvent was removed in vacuum. The mixture was dissolved in EtOAc (50 mL)

and washed by EDTA solution (0.5%, 10 mL), water (3x10 mL) and brine (1x10 mL). The

mixture was dried over anhydrous Na2SO4. The solvent was evaporated and the product

was purified by column chromatography (EtOH:CHCl3).

Examples of A

Red solid, 220 mg (64%); mp = over 250°С with decomposition; 1H NMR (700 MHz,

DMSO) δ 11.96 (bs, 1H), 10.16 (bs, 1H), 8.53 (s, 1H), 8.01 (d, J = 15.6 Hz, 1H), 7.99 (d, J =

8.6 Hz, 2H), 7.97 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.16

(t, J = 7.8 Hz, 1H), 6.75 (d, J = 15.6 Hz, 1H), 6.70 (s, 1H), 6.24 (d, J = 8.6 Hz, 2H), 3.25 (s,

3H); 13C NMR (176 MHz, DMSO) δ 170.1, 157.8, 157.6, 137.5, 134.1, 131.9, 124.9, 123.7,

122.6, 120.9, 120.3, 118.1, 116.6, 116.3, 116.2, 113.4, 112.4, 106.9, 26.2; HRMS (ESI) m/z:

344,1406 found (calcd. for C21H18N3O2, [M+H]+ 344.1399) 11.

Orange solid, 370 mg (77%); mp = 203-205°С; 1H NMR (700 MHz, DMSO) δ 10.44 (bs,

1H), 10.17 (bs, 1H), 8.19 (d, J = 8.6 Hz, 2H), 8.15 (s, 2H), 7.83 (d, J = 15.7 Hz, 1H), 7.19 (d,

J = 15.7 Hz, 1H), 6.95 (s, 1H), 6.87 (d, J = 8.6 Hz, 2H), 3.26 (s, 3H); 13C NMR (176 MHz,

DMSO) δ 169.9, 159.7, 158.6, 152.0, 136.9, 136.7, 134.5, 132.1, 130.0, 125.8, 125.7, 115.8,

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113.8, 112.2, 26.4; HRMS (ESI) m/z: 478.9424 found (calcd. for C19H15Br2N2O3, [M+H]+

478.9429).

Orange solid, 340 mg (73%); mp = over 250°С with decomposition; 1H NMR (700 MHz,

DMSO) δ 11.58 (bs, 1H), 10.44 (bs, 1H), 10.14 (bs, 1H), 8.10 (d, J = 8.7 Hz, 2H), 7.87 (s,

2H), 7.43 (d, J = 16.4 Hz, 1H), 7.01 (d, J = 16.4 Hz, 1H), 6.88 (s, 1H), 6.84 (d, J = 8.7 Hz,

2H);13C NMR (176 MHz, DMSO) δ 171.2, 159.6, 158.7, 151.9, 138.3, 136.5, 134.2, 131.4,

129.8, 125.7, 125.4, 117.3, 115.8, 112.3; HRMS (ESI) m/z: 464.9261 found (calcd. for

C18H13Br2N2O3, [M+H]+ 464.9272).


1H), 8.19 (d, J = 8.6 Hz, 2H), 7.95 (d, J = 15.9 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.28 (d, J =

8.0 Hz, 2H), 7.19 (d, J = 15.9 Hz, 1H), 6.95 (s, 1H), 6.89 (d, J = 8.6 Hz, 2H), 3.27 (s, 3H),

2.36 (s, 3H); 13C NMR (176 MHz, DMSO) δ 170.0, 159.6, 158.9, 139.9, 139.5, 136.9, 134.4,

132.5, 129.5, 128.2, 125.8, 125.6, 115.8, 112.9, 26.3, 21.0; HRMS (ESI) m/z: 319.1440

found (calcd. for C20H19N2O2, [M+H]+ 319.1447).


1H), 8.22-8.15 (m, 3H), 7.95-7.90 (m, 1H), 7.36-7.26 (m, 3H), 7.11 (d, J = 15.8 Hz, 1H), 6.98

(s, 1H), 6.87 (d, J = 8.7 Hz, 2H), 3.28 (s, 3H), 2.54 (s, 3H); 13C NMR (176 MHz, DMSO) δ

169.9, 159.7, 158.8, 137.2, 136.8, 136.6, 134.4, 133.9, 130.7, 129.7, 126.5, 126.3, 126.0,

125.8, 115.9, 115.1, 26.4, 19.3; HRMS (ESI) m/z: 319.1438 found (calcd. for C20H19N2O2,

[M+H]+ 319.1447).

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Orange solid, 285 g (86%); mp = 223-227°С; 1H NMR (700 MHz, DMSO) δ 10.15 (bs,

1H), 8.18 (d, J = 8.6 Hz, 2H), 7.93 (d, J = 15.8 Hz, 1H), 7.65 (bs, 1H), 7.58 (bd, J = 7.5 Hz,

1H), 7.23 (d, J = 7.5 Hz, 1H), 7.15 (d, J = 15.8 Hz, 1H), 6.95 (s, 1H), 6.87 (d, J = 8.6 Hz, 2H),

3.27 (s, 3H), 2.29 (s, 3H), 2.27 (s, 3H); 13C NMR (176 MHz, DMSO) δ 169.9, 159.6, 158.9,

139.7, 138.8, 136.9, 136.8, 134.4, 132.8, 130.0, 129.2, 125.9, 125.8, 125.4, 115.8, 112.7,

26.3, 19.4, 19.2; HRMS (ESI) m/z: 333.1696 found (calcd. for C21H21N2O2, [M+H]+

333.1603).


1H), 8.19 (d, J = 8.6 Hz, 2H), 7.97 (d, J = 15.9 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.53 (d, J =

8.4 Hz, 2H), 7.25 (d, J = 15.9 Hz, 1H), 6.98 (s, 1H), 6.87 (d, J = 8.6 Hz, 2H), 3.27 (s, 3H); 13C

NMR (176 MHz, DMSO) δ 169.9, 159.8, 158.5, 137.9, 136.9, 134.5, 134.4, 134.2, 129.9,

128.9, 126.2, 125.8, 115.9, 114.9, 26.4; HRMS (ESI) m/z: 339.0894 found (calcd. for

C19H16ClN2O2, [M+H]+ 339.0900).

References

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