their mutated derivatives the unusual metal ion binding ability of … · 2016. 2. 17. · e-mail:...
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Supporting Information for
"The unusual metal ion binding ability of histidyl tags and
their mutated derivatives"
Davide Brasili,a Joanna Watly, b Eyal Simonovsky, cd Remo Guerrini,a Nuno A. Barbosa, b
Robert Wieczorekb, Maurizio Remellia, Henryk Kozlowskib* and Yifat Millercd*
a Department of Chemical and Pharmaceutical Sciences, University of Ferrara, via Fossato di Mortara 17, I-44121 Ferrara, Italy. E-mail: [email protected]
b Department of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland. E-mail: [email protected]
c Department of Chemistry and dIlse Katz Institute for Nanoscale Science and Technology, Ben Gurion University of the Negev, Beer-Sheva 84105, Israel. E-mail: [email protected]
Electronic Supplementary Material (ESI) for Dalton Transactions.This journal is © The Royal Society of Chemistry 2016
Table S1: Complex-formation constants for the ligand L1 with Cu(II), at T = 298 °K and I = 0.1 mol·dm−3 (KCl). Standard deviations on the last figure in parentheses.
Species Log pKa[CuLH3]2+ 25.68 (6) 4.3[CuLH2]+ 21.38 (3) 5.19[CuLH] 16.19 (4) 6.82[CuL]- 9.37 (6) 7.87
[CuLH-1]2- 1.50 (7) 8.25[CuLH-2]3- -6.75 (8) 10.05[CuLH-3]4- -16.8 (1) -
Table S2: Complex-formation constants for the ligand L2 with Cu(II), at T = 298 °K and I = 0.1 mol·dm−3 (KCl). Standard deviations on the last figure in parentheses.
Species Log pKa[CuLH4]3+ 32.26 (5) 4.44[CuLH3]2+ 27.82 (4) 4.58[CuLH2]+ 23.24 (3) 5.91[CuLH] 17.33 (4) 6.43[CuL]- 10.90 (5) -
[CuLH-2]3- -5.16 (6) 9.40[CuLH-3]4- -14.56 (8) -
Table S3: Spectroscopic parameters at different pH values for the system Cu(II)/L1; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 1.1.1.
UV-Vis CD EPR
pH Species /nm ε/M-1cm-1 λ/nm Δε/M-1 cm-1 AII (G) gII
4.5 [CuLH3]2+ [CuLH2]+ 663 41.1 254 0.15 164 2.32
5.0 [CuLH2]+ [CuLH] 635 67.2 580
250-0.131.36 166 2.28
5.6 [CuLH2]+ [CuLH] 622 80.8 586
252-0.212.17
6.2 [CuLH] [CuL]- 610 87.7 570245
-0.252.89 173 2.27
7.1 [CuL]- [CuLH] [CuLH-1]2- 608 90 570
250-0.253.11 186 2.25
7.5 [CuL]- [CuLH][CuLH-1]2- 603 92
677557457341250
0.10-0.300.06-0.133.47
8.0 [CuL]- [CuLH-1]2- [CuLH-2]3- 596 106
640548478336250
0.21-0.440.99-1.074.77
188 2.23
8.6 [CuLH-1]2- [CuLH-2]3- 577 123
633551474335252
0.44-0.460.16-1.725.41
9.1 [CuLH-1]2-
[CuLH-2]3- 562 140
638551474335254
0.51-0.460.16-1.725.59
188 2.23
9.6 [CuLH-2]3-
[CuLH-3]4- 558 130
635531402337254
0.80-0.260.16-1.245.96
10.3 [CuLH-2]3-
[CuLH-3]4- 530 128
631487402345260
0.96-0.700.06-0.536.12
186 2.22
11.2 [CuLH-3]4- 521 135
626486402318294258
1.37-1.180.020.76-0.196.78
193 2.19
Table S4: Spectroscopic parameters at different pH values for the system Cu(II)/L2; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 1.1.1.
UV-Vis CD EPR
pH Species /nm ε/M-1cm-1 λ/nm Δε/M-1 cm-1 AII (G) gII
4.7[CuLH4]3+ [CuLH3]2+ [CuLH2]+
659 36.1 252 0.63 164 2.31
5.2 [CuLH3]2+ [CuLH2]+ 639 52.0 249 1.30 169 2.28
6.2[CuLH2]+
[CuLH] [CuL]-
607 68.1 246 1.75 - -
6.7 [CuLH] [CuL]- 607 69.3 246 2.00 173 2.27
7.2 [CuL]- 602 73.4 247 2.44
7.7 [CuL]- [CuLH-2]3- 588 82.6
635557251
0.07-0.192.75
182 2.25
8.4 [CuL]- [CuLH-2]3- 579 89.8
636549476335247
0.20-0.390.09-1.214.41
186 2.21
8.8 [CuLH-2]3- [CuLH-3]4- 556 100.6
632548402336249
0.40-0.580.02-1.765.91
- -
10.0 [CuLH-2]3- [CuLH-3]4- 537 105.06
624517404339256
0.83-0.420.10-0.966.37
188 2.20
10.9 [CuLH-3]4- 522 108.5
624485353316294259
1.18-0.99-0.240.700.256.46
191 2.20
11.5 [CuLH-3]4- 518 109.3
626489358320294261
1.24-1.13-0.160.87-0.166.84
- -
Table S5: Metal-ligand interactions (L1a and L1b complexes).
X Cu-X [Å]L1a
His7 1.832His11 1.835
L1bHis5 2.440His7 1.849His11 1.841
Table S6: Metal-ligand interactions (L2 complex).
X Cu-X [Å]L2
His4 1.875His6 2.130His10 1.901
Table S7: Hydrogen bonds of L1a and L1b complexes. Fragments marked with star supply hydrogen bond with atoms form side chains.
Residue H..PA[Å]
PD-H..PA[deg]
Fragment HB type
L1aD2..H11 1.604 162.3 C=O*..H-N*D2..H11 1.637 170.6 C=O*..H-NA6..A8 2.195 157.8 N-H..O=C 3-10 helixA6..H9 2.105 151.3 C=O..H-N*Ac..D2 2.005 153.8 C=O(Ac)..H-NAc..D2 1.828 168.4 C=O(Ac)..H-N
L1bH5...A12 1.878 166.2 N-H*..O=CE1..H11 1.703 163.0 N-H..O=C*H7..H9 2.461 162.7 C=O..H-N 3-10 helixA6..A8 1.905 167.1 C=O..H-N 3-10 helixD2..D3 1.950 168.6 C=O*..H-NAc..A6 1.847 162.5 C=O(Ac)..H-N
Table S8: Hydrogen bonds of S2 complex. Fragments marked with star supply hydrogen bond with atoms form side chains.
Residue H..PA[Å]
PD-H..PA[deg]
Fragment HB type
S2E1..D3 1.987 150.7 N-H..O=C*D3..H4 1.180 153.6 C=O*..H-N*D3..A5 1.679 167.9 O=C..N-H 3-10 helixH4..H6 2.001 165.6 O=C..N-H 3-10 helixA5..A7 1.788 165.0 O=C..N-H 3-10 helixH6..H8 2.181 164.9 O=C..N-H 3-10 helixA7..A9 2.095 156.2 O=C..N-H 3-10 helix
A7..NH2 2.079 158.8 O=C..N-H2
H4..G13 1.704 162.3 N-H*..O=CH10..A11 1.670 154.2 N-H..O=C
Table S9: Conformational energies and populations obtained from the GBMV and Monte-Carlo calculations.
Peptide Averaged conformational energy
[kcal/mol]
Standard error of the averaged conformational energy values
[kcal/mol]
Populations (%)
L1a -819.9 0.7 27
L1b -861.0 0.6 73
2 3 4 5 6 7 8 90
10
20
30
40
50
60
70
80
90
100
% fo
rmat
ion
rela
tive
to [L
]
pH
L3-
LH3+6 LH2+
5
LH+4
LH3
LH-2
LH2-
LH4+7
Figure S1. Species distribution diagram for the protonation equilibria of the ligand L1; C°L = 6·10−4 mol·dm−3.
2 3 4 5 6 7 8 90
10
20
30
40
50
60
70
80
90
100%
form
atio
n re
lativ
e to
[L]
pH
LH5+8
LH4+7
LH3+6
LH2+5
LH+4
LH3
LH-2
LH2-
L3-
Figure S2. Species distribution diagram for the protonation equilibria of the ligand L2; C°L = 6·10−4 mol·dm−3.
460.5
562.3
604.8
648.2
690.3
721.2
879.2926.4
1087.3
200 400 600 800 1000 1200 1400 m/z0
2
4
6
9x10Intens.
Figure S3. ESI-MS spectrum of the system Cu(II)/L1; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; positive ion mode.
688.3
718.7
1377.5
1475.4
400 600 800 1000 1200 1400 1600 m/z0.0
0.5
1.0
1.5
2.0
2.5
8x10Intens.
Figure S4 ESI-MS spectrum of the system Cu(II)/L1; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; negative ion mode.
1440.5
1441.0
1441.5
1442.0
1442.5
1443.0
1443.5
1444.5
1445.51446.5
2014_04_04_Asia_Davide_CuL1_21_pos_do_kal_000001.d: +MS
1440.5
1441.5 1442.5
1443.5
1444.51445.5
2014_04_04_Asia_Davide_CuL1_21_pos_do_kal_000001.d: CuC56H77N22O20, M ,1440.500.00
0.25
0.50
0.75
1.00
1.25
1.508x10
Intens.
0
500
1000
1500
2000
2500
1440 1441 1442 1443 1444 1445 1446 1447 1448 m/z
Figure S5. Experimental (up) and simulated (down) ESI-MS spectrum for the complex [CuLH2]+ (CuC56H77N22O20, MW = 1440.6) in the system Cu(II)/L1; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; positive ion mode.
718.7 719.2
719.7
720.2
720.7721.2 721.7
2014_04_04_Asia_Davide_CuL1_21_low_neg_000001.d: -MS
718.7
719.2 719.7
720.2
720.7721.2
2014_04_04_Asia_Davide_CuL1_21_low_neg_000001.d: C56H74N22O20Cu, M ,1437.470.0
0.2
0.4
0.6
0.8
8x10Intens.
0
500
1000
1500
2000
2500
718.5 719.0 719.5 720.0 720.5 721.0 721.5 722.0 m/z
Figure S6. Experimental (up) and simulated (down) ESI-MS spectrum for the complex [CuLH-1]2- (CuC56H74N22O20, MW = 1437.6) in the system Cu(II)/L1; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; negative ion mode.
1500.41500.9
1501.4
1501.9
1502.4
1502.9
1503.4
1503.9
1504.4
1504.9
1505.4
1505.9
1506.41507.4
1508.4
2014_04_04_Asia_Davide_CuL1_21_pos_do_kal_000001.d: +MS
1501.4
1502.4
1503.4
1504.4
1505.4
1506.4
1507.4
2014_04_04_Asia_Davide_CuL1_21_pos_do_kal_000001.d: C56H75N22O20Cu2, M ,1501.410
2
4
6
7x10Intens.
0
500
1000
1500
2000
2500
1500 1502 1504 1506 1508 1510 m/z
Figure S7. Experimental (up) and simulated (down) ESI-MS spectrum for the complex [Cu2L]+ (Cu2C56H75N22O20, MW = 1501.4) in the system Cu(II)/L1; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; positive ion mode.
524.5
590.2
637.8668.7
723.3
754.3
785.2
817.2
200 400 600 800 1000 1200 1400 m/z0
2
4
6
8
8x10Intens.
+MS
Figure S8. ESI-MS spectrum of the system Cu(II)/L2; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; positive ion mode.
401.7
721.3
1443.5
200 400 600 800 1000 1200 1400 1600 1800 2000 m/z0.0
0.5
1.0
1.5
8x10Intens.
Figure S9. ESI-MS spectrum of the system Cu(II)/L2; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; negative ion mode.
753.8754.3
754.8
755.3
755.8756.3
2014_03_25_Asia_Davide_Lig2Cu_pos_do_kal_000001.d: +MS
0.0
0.5
1.0
1.5
9x10Intens.
754.0 754.5 755.0 755.5 756.0 756.5 757.0 m/z
753.8
754.3 754.8
755.3
755.8756.3
2014_03_25_Asia_Davide_Lig2Cu_pos_medium_000001.d: C59H80N24O20Cu, M ,1507.53
0
500
1000
1500
2000
2500
754.0 754.5 755.0 755.5 756.0 756.5 757.0 m/z
Figure S10. Experimental (up) and simulated (down) ESI-MS spectrum for the complex [CuLH3]2+ (CuC59H80N24O20, MW = 1507.6) in the system Cu(II)/L2; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; positive ion mode.
803.2803.7
804.2804.7
805.2
805.4
805.7
806.2 806.7806.9
807.2
807.3
807.6
2014_04_04_Asia_Davide_CuL2_21_pos_do_kal_000001.d: +MS
0.0
0.5
1.0
1.5
7x10Intens.
803.0 803.5 804.0 804.5 805.0 805.5 806.0 806.5 807.0 807.5 m/z
803.2
803.7
804.2
804.7
805.2
805.7
806.2
2014_03_25_Asia_Davide_Lig1Cu_pos_medium_000001.d: C59H77N24O20Cu2K, M ,1606.40
0
500
1000
1500
2000
2500
803.0 803.5 804.0 804.5 805.0 805.5 806.0 806.5 807.0 807.5 m/z
Figure S11. Experimental (up) and simulated (down) ESI-MS spectrum for the complex [Cu2L]K2+ (Cu2C59H77N24O20K, MW = 1606.4) in the system Cu(II)/L2; C°Cu(II) = 5·10−4 mol·dm−3; M/L ratio = 2.1; pH = 5; positive ion mode.
2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700
Cu(II) - Ac-EDDAHAHAHAHAG-NH2
pH 8.18pH 6.14
pH 3.15
pH 5.15pH 4.16
pH 9.16
pH 11.11pH 10.10
Magnetic field [G]
pH 7.16
pH AII [G] gII
3.15 120.6 2.424.16 164.4 2.325.15 166.5 2.286.14 173.1 2.277.16 186.3 2.258.18 188.3 2.239.11 188.5 2.23
10.10 186.3 2.2211.11 192.8 2.19
Figure S12. EPR spectra at different pH for the system Cu(II)/L1; C°Cu(II) = 1·10−3 mol·dm−3; M/L ratio = 1.1.1.
2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700
pH 10.50pH 9.50pH 8.67
pH 3.81pH4.77pH 5.50pH 6.67pH 7.40
Magnetic field [G]
pH 3.11
Cu(II) - Ac-EDDHAHAHAHAHG-NH2
pH A II[G] g II3.11 120.6 2.413.81 - 2.324.77 164.4 2.305.50 168.8 2.286.67 173.2 2.277.40 182.0 2.258.67 186.0 2.219.50 188.5 2.20
10.50 190.7 2.20
Figure S13. EPR spectra at different pH for the system Cu(II)/L2; C°Cu(II) = 1·10−3 mol·dm−3; M/L ratio = 1.1.1.
Figure S14 DSSP calculations for each residue along the peptides along the time of the simulations.
Figure S15 Populations of the simulated peptides L1a and L1b models. The populations had been estimated via Monte Carlo simulations.
Figure S16 The Cu(II)-Nε (His) atom and Cu(II)-O (carboxyl group) atoms distance distribution for each His residue obtained from MD simulations for the structures L1a, L1b and L2. The vertical lines within each box represent the median distance values.
L2
Figure S17. Synthetic network of hydrogen bonds in Complexes L1a, L1b and L2.
L1a
L1b
(a)
3 4 5 6 7 8 9 10 110
10
20
30
40
50
60
70
80
90
100
% for
matio
n rela
tive t
o [Cu
(II)]
pH
Cu(II) - Ac-THHHHAHGG-NH2
Cu(II) - Ac-EDDAHAHAHAHAG-NH2
free Cu(II)
(b)
3 4 5 6 7 8 9 10 110
10
20
30
40
50
60
70
80
90
100
% fo
rmtio
n re
lative
to [C
u(II)
]
pH
free Cu(II)
Cu(II) - Ac-EDDHAHAHAHAHG-NH2
Cu(II) - Ac-THHHHAHGG-NH2
Figure S18. Competition plot for a ternary solution containing equimolar concentrations (1 mM) of: (a) Cu(II), L1 and Ac- THHHHAHGG-NH2 (H. pylori); (b) Cu(II), L2 and Ac- THHHHAHGG-NH2 (H. pylori).
(a)
3 4 5 6 7 8 9 10 110
10
20
30
40
50
60
70
80
90
100
% fo
rmati
on re
ltive t
o [Cu
(II)]
pH
Cu(II) - Ac-PVHTGHMGHIGHTGHTGHTGSSGHG-NH2
Cu(II) - Ac-EDDAHAHAHAHAG-NH2
free Cu(II)
(b)
3 4 5 6 7 8 9 10 110
10
20
30
40
50
60
70
80
90
100
Cu(II) - Ac-PVHTGHMGHIGHTGHTGHTGSSGHG-NH2
% fo
rmati
on re
lative
to [C
u(II)
]
pH
Cu(II) - Ac-EDDHAHAHAHAHG-NH2
free Cu(II)
Figure S19. Competition plot for a ternary solution containing equimolar concentrations (1 mM) of: (a) Cu(II), L1 and Ac- PVHTGHMGHIGHTGHTGHTGHTGSSHG-NH2 (zp-PrP63-87); (b) Cu(II), L2 and Ac- PVHTGHMGHIGHTGHTGHTGHTGSSHG-NH2 (zp-PrP63-87).
(a)
3 4 5 6 7 8 9 10 110
10
20
30
40
50
60
70
80
90
100
% fo
rmat
ion re
lative
to [C
u(II)
]
pH
free Cu(II)
Cu(II) - Ac-HHHHHH-NH2
Cu(II) - Ac-EDDAHAHAHAHAG-NH2
(b)
3 4 5 6 7 8 9 10 110
10
20
30
40
50
60
70
80
90
100
Cu(II) - Ac-EDDHAHAHAHAHG-NH2
Cu(II) - Ac-HHHHHH-NH2
free Cu(II)
% fo
rmat
ion re
lative
to [C
u(II)
]
pH
Figure S20. Competition plot for a ternary solution containing equimolar concentrations (1 mM) of: (a) Cu(II), L1 and Ac-HHHHHH-NH2 (His6-tag) ; (b) Cu(II), L2 and Ac-HHHHHH-NH2 (His6-tag).