Henryk Kozlowski Faculty of Chemistry

University of Wroclaw

CAGLIARI, SEPTEMBER, 2013

Katherine H. Thompson and Chris Orvig, Science 300, 936 (2003)

Barry J. Marshall

J. Robin Warren

Barry J. Marshall

J. Robin Warren

Urease makes envi-ronment around the bacteria more basic

Hydrogenase plays a vital role in anaerobic metabolism

Urease and hydrogenase accessory proteins, involved in the homeostasis of Ni2+

How can Helicobacter pylori survive in adverse conditions of our stomach?

CO(NH2)2 + H2O→ 2 NH3 + CO2

The active site of urease contains two nickel atoms (green) bridged by a carboxylated lysine and a hydroxyl group.

The urease active site

Eric L. Carter,a Nicholas Flugga, Jodi L. Boer,b Scott B. Mulrooneya and Robert P. Hausinger, Metallomics, 2009, 1, 207-221

Urease maturation

D. Witkowska, M. Rowińska-Żyrek, G. Valensin and H. Kozłowski. Coord.Chem.Rev., 2012, 256, 133-148.

The active site of [NiFe] hydrogenase

W. Zhu, A. C. Marr, Q. Wang, F. Neese, D. J. E. Spencer, A. J. Blake, P. A. Cooke, C. Wilson, and M. Schroder, PNAS, 2005, 18280–18285

Fe2+

HycE

HypC

HypC

HypD

HypE

HypF

HypA

HypB

SlyD

ATP

ADP+Pi

ATPCP

AMP+PPi

HycE

Ni2+GTP

HycEHypC

HypC

GDP+Pi

HycE

HycI

HycI

Fe

Ni

S SFe

Ni

S S

Fe

Ni

S S

Fe

Fe2+

HycE

HypC

HypC

HypD

HypE

HypF

HypA

HypB

SlyD

ATP

ADP+Pi

ATPCP

AMP+PPi

HycE

Ni2+GTP

HycEHypC

HypC

GDP+Pi

HycE

HycI

HycI

Fe

Ni

S SFe

Ni

S S

Fe

Ni

S S

Fe

D. Witkowska, M. Rowińska-Żyrek, G. Valensin and H. Kozłowski. Coord.Chem.Rev., 2012, 256, 133-148.

Hydrogenase maturation

- Metronidazole 500 mg + Amoxycycline 1 g + PPI

- Klatromycine 500 mg +

Metronidazol 500 mg + PPI - Klatromycine 500 mg +

Amoxycycline 1 g + PPI

PPI: omeprazole, lansoprazole, rabeprazole, esomeprazole, pantoprozole

RBS, rantidine bismuth subcitrate (Tritec, Pylorid) Bismuth(III) compounds CBS, colloidal bismuth subcitrate (De-Nol) BSS, bismuth subsalicate

Getting rid of H. pylori

M. Rowińska-Żyrek, D. Valensin, Ł. Szyrwiel, Z. Grzonka, H. Kozłowski, Dalton Trans., 2009, 9131-9140

Species log β logK

HL

H2L

H3L

H4L

H5L

H6L

10.75 (2)

21.02 (1)

30.13 (2)

38.57 (2)

46.40 (2)

50.13 (3)

10.75 Lys

10.25 Cys

9.11 Cys

8.44 Cys

7.83 Cys

3.75 Asp

BiH2L 52.29 (8)

BiHL 48.51 (4) 3.78

BiL 39.32 (5) 9.19 2 4 6 8 10

0

20

40

60

80

100

BiL

BiHLBiH

2L

Ac-GCASCDNCRAAKK-NH2 : Bi

3+ = 2:1

% fo

rma

tio

n r

ela

tive

to

Bi3

+

pH

free Bi3+

The complicated relationship between bismuth and protein fragments:

• Cysteine thiol sulfurs are the only anchoring sites for bismuth • Extremaly stable complexes are formed already at pH < 1

Competition plot of Ac-CGC-NH2, MDPETCPCP-NH2, Ac-CHCH-NH2 and Ac-ACCHDHKKH-NH2 with Bi3+ (1:1:1:1:2)

• Coordination of Bi3+ to the CXC sequence – much stronger than the coordination to the CC sequence • Involvement of histidines in the binding of Bi3+ enhances the stability of the complex

M. Rowińska-Żyrek, D. Valensin, L. Szyrwiel, Z. Grzonka and H. Kozlowski, Dalton Trans., 2009, 9131 M. Rowińska-Żyrek, D. Witkowska, D. Valensin, W. Kamysz and H. Kozlowski, Dalton Trans, 2010, 39 , 5814.

2 4 6 8

0

20

40

60

80

100

Bi3+

-[Ac-CGC-NH2]2 Bi

3+-Ac-ACCHDHKKH-NH

2

Bi3+

-MDPETCPCP-NH2

Bi3+

-Ac-CHCH-NH2

% fo

rma

tio

n r

ela

tive

to

Bi3

+

pH

free Bi3+

How strong are Bi3+ - imidazole interactions?

2 4 6 8 10

0

10

20

30

40

50

60

70

80

90

100

Bi3+

Bi3+

-Ac-CHCH-NH2

Bi3+

-MPGCPCPGCG-NH2

Bi3+

: MPGCPCPGCG-NH2 : Ac-CHCH-NH

2 = 1:1:1

% fo

rma

tio

n r

ela

tive

to

[B

i3+]

pH

Potocki S., Rowińska-Żyrek M., Valensin D., Krzywoszyńska K, Witkowska D., Łuczkowski M, Kozłowski H., Inorg. Chem, 2011

2 4 6 8 10

0

10

20

30

40

50

60

70

80

90

100

Bi-Ac-CCSTSDSHHQ-NH2

Bi-MDPETCPCP-NH2

Bi-Ac-GCASCDNARAAKK-NH2

Ac-GCASCDNARAAKK-NH2 : MDPETCPCP-NH

2 : Ac-CCSTSDSHHQ-NH

2 : Bi

3+ = 1:1:1

% fo

rma

tio

n r

ela

tive

to

[B

i3+]

pH

What is the impact of the distance between Cys residues in peptide fragments on the stability of the complexes they form with Bi3+ ions?

CXXC

CXC

CC

Rowińska-Żyrek M., Witkowska D., Bielinska S., Kamysz W. and Kozlowski H., Dalton Trans., 2011, 40, 5604-5610.

2 4 6 8 10

0

20

40

60

80

100

Bi-Ac-GCASCDNARAAKK-NH2

Bi-Ac-GCASCDNCRAAKK-NH2

Bi-Ac-GCASCDNCRACKK-NH2

Ac-GCASCDNCRACKK-NH2 : Ac-GCASCDNCRAAKK-NH

2 : Ac-GCASCDNARAAKK-NH

2 : Bi

3+ = 1:1:1:1

% fo

rma

tio

n r

ela

tive

to

[B

i3+]

pH

What is the correlation between the intensity of charge transfer bands and the amount of sulfur atoms bound to the Bi3+ ion?

Amount of

bound Cys

ε [M-1 cm-1]

2 Cys 3500

3 Cys 4500

4 Cys 5200

K. Krzywoszynska, M. Rowinska-Zyrek, D. Witkowska, S. Potocki, M. Luczkowski, H. Kozłowski Dalton Trans., 2011, 40, 10434-10439

91GSCCHTGNHDHKHAKEHEACCHDHKKH118

Cun et al., J. Biol. Chem. 2008, 283, 22, p. 15142

300 400 500

-10

0

10

NiLBi0,6

NiLBi0,45

NiLBi0,3

NiLBi0,15

NiL

[nm]

CD spectra of Ni2+ complexes with the B-domain peptide at pH6, titrated with Bi3+ ions in a step of 0.15 molar equivalents. Final Bi3+ : Ni2+ : L ratio = 1 : 1 : 2.

Bi3+ is able to displace Ni2+ from it’s binding sites:

M. Rowińska-Żyrek, D. Witkowska, D. Valensin, H. Kozlowski, Dalton Trans. 2010, 39 , 5814.

M. Rowińska-Żyrek, D. Witkowska, D. Valensin, H. Kozlowski, Dalton Trans. 2010, 39 , 5814.

W. Xia, H. Li, K. H. Sze and H. Sun, J. Am. Chem. Soc. 131 (2009) 10031–10040.

Metallochaperone HypA

Communication between the zinc and nickel sites in dimeric HypA

Herbst R.W.; Perovic I.; Martin-Diaconescu V.; O’Brien K.; Chivers P.T.; Pochapsky S.S.; Pochapsky T.C.; Maroney M.J.; J. Am. Chem. Soc. 2010, 132 (30), 10338–10351

W. Xia, H. Li, K. H. Sze and H. Sun, J. Am. Chem. Soc. 131 (2009) 10031–10040.

Metallochaperone HypA

Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2

HypA loop sequence - residues 71-96

764.3

746.3

749.8

755.6

763.9765.1

766.1 768.8770.9

745 750 755 760 765 770 775 m/z0.0

0.2

0.4

0.6

0.8

1.0

1.2

8x10

Intens.

ZnL4+

L4+

NaL4+

Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2 + 1 eq. Zn(II)

763.6

749.8

755.3

745 750 755 760 765 770 775 m/z0

1

2

3

4

5

6

8x10

Intens.

NiL4+

L4+

NaL4+

Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2 + 1 eq. Ni(II)

ESI-MS spectra of a system containing Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2,

with Zn2+ (A) and Ni2+ ions (B) in a 1 : 1 : 1 stoichiometry

M. Rowinska-Zyrek, S. Potocki, , D. Witkowska, D.Valensin H. Kozlowski, Dalton Trans., 42, 6012-6020, (2013)

4 6 8 10

0

20

40

60

80

100

% fo

rma

tion

re

lativ

e to

lig

an

d

pH

ZnL

NiL

Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2 : Zn

2+ : Ni

2+ = 1 : 1 : 1

A competition plot of Ni2+ and Zn2+ complexes with the

Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2 fragment

M. Rowinska-Zyrek, S. Potocki, , D. Witkowska, D.Valensin H. Kozlowski, Dalton Trans. 42, 6012-6020, (2013)

conserved –CXXC- motifs

Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2

Asp

Cys Ser

His

Val Ni

Communication between the zinc and nickel sites in dimeric HypA

Herbst R.W.; Perovic I.; Martin-Diaconescu V.; O’Brien K.; Chivers P.T.; Pochapsky S.S.; Pochapsky T.C.; Maroney M.J.; J. Am.

Chem. Soc. 2010, 132 (30), 10338–10351

Types of binding modes in Zn2+ complexes with the

Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2 fragment

M. Rowinska-Zyrek, D. Witkowska, D.Valensin, S. Potocki, H. Kozlowski, Dalton Trans., 42, 6012-6020, (2013)

4 6 8 10

0

20

40

60

80

100

Zn2+

ZnHL

ZnLZnH

2L

ZnH3L

ZnH4L

ZnH5L

% fo

rma

tio

n r

ela

tive to

[Z

n2

+]

pH

{3S-,Nim} {4S-}

Metal ions + Hpn

R. Ge, Y. Zhang, X. Sun, R. Watt, Q-Y. He, J-D. Huang, D. Wilcox, H. Sun, J. Am. Chem. Soc., 2006, 128 (35), 11330

MAHHEEQHGG HHHHHHHTHH HHYHGGEHHH

HHHSSHHEEG CCSTSDSHHQ EEGCCHGHHE

…a specific sequence of string of six to nine histidine residues

…used in vectors for productions of recombinant proteins Recombinant protein with a six histidine or poly-His tag connected to N- or C-terminus

Expressed His-tagged proteins can be purified and

detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, zinc and copper, under specific buffer conditions.

The chelators most commonly used as ligand for IMAC:

o nitrilotriacetic acid (NTA)

o iminodiacetic acid (IDA)

Elution by:

o adding free imidazole

o by EDTA

o by reducing the pH

Model of the interaction between residues in the His-tag and metal ion in tetradentate IMAC ligand (NTA).

A. Trampuz, M. Jereb, I. Muzlovic and R. Prabhu, Crit.Care, 2003, 7(4), 315.

4 6 8 10

0

20

40

60

80

100

free L

Ni2+

-Ac-CCSTSDSHHQ-NH2

Zn2+

-Ac-CCSTSDSHHQ-NH2

% fo

rma

tio

n r

ela

tive

to

L

pH

The Ni2+ complex of Ac-CCSTSDSHHQ-NH2 peptide

from H.pylori Hpn protein is much more stable than that of zinc

M. Rowińska-Żyrek, D. Witkowska, S. Bielińska, W. Kamysz, H. Kozłowski. Dalton Trans., 2011, 40, 5604-5610

MAHHEEQHGG HHHHHHHTHH HHYHGGEHHH

HHHSSHHEEG CCSTSDSHHQ EEGCCHGHHE

4 6 8 10

0

20

40

60

80

100

% fo

rma

tion

re

lativ

e to

L

free L

Zn-Ac-EEGCCHGHHE-NH2

Ni2+

-Ac-EEGCCHGHHE-NH2

pH

The Ni2+ complex of Ac-EEGCCHGHHE-NH2 peptide from H.pylori Hpn protein is much more stable

than that of zinc

M. Rowińska-Żyrek, D. Witkowska, S. Bielińska, W. Kamysz, H. Kozłowski. Dalton Trans., 2011, 40, 5604-5610

MAHHEEQHGG HHHHHHHTHH HHYHGGEHHH

HHHSSHHEEG CCSTSDSHHQ EEGCCHGHHE

300 400 500 600 700 800

-20

-10

0

10

20

(mol

-1cm

-1)

pH4

pH7-pH9

Ni2+

-Ac-EEGCCHGHHE-NH2

pH61

[nm]

pH5

300 400 500 600 700 800

-50

-40

-30

-20

-10

0

10

20

30

40

50

Ni2+

-Ac-CC-NH2

pH7-pH10

pH6

pH5,3/m

olxc

m[nm]

pH4

M. Rowińska-Żyrek, D. Witkowska, S. Bielińska, W. Kamysz, H. Kozłowski. Dalton Trans., 2011, 40, 5604-5610

CD spectra of Ni2+complexes of Ac-CC-NH2 and Ac-EEGCCHGHHE-NH2

peptides

X

NH2CH

CH2

O

S

N

CHCH2

O

S

NH2

Ni

MAHHEQQQQQQA-NH2 (Hpn-like) MAHHEEQHG-NH2 (Hpn) Ac-EEGCCHGHHE-NH2 (Hpn) Ac-THHHHYHGG-NH2 (Hpn) Ac-ACCHDHKKH-NH2 (HspA)

Copper and nickel binding to fragments

of Helicobacter pylori proteins

4 6 8 10

0

20

40

60

80

100

DAH-NH2 : Ac-ACCHDHKKH-NH

2 : Ni

2+= 1:1:1

free Ni2+

Ni2+

-Ac-ACCHDHKKH-NH2

% fo

rmat

ion

rela

tive

to N

i2+

pHM. Rowinska-Zyrek, D. Witkowska, D. Valensin, W. Kamysz and H. Kozlowski, Dalton Trans, 2010, 39 , 5814 - 5826 P. Mlynarz, D. Valensin, K. Kociolek, J. Zabrocki, J. Olejnik, H. Kozlowski, New J. Chem., 2002, 26, p.264

The distribution of Ni2+ between DAH-NH2 and Ac-ACCHDHKKH-NH2 peptides

HN

CH

C

H2C

O

S

NCH

C

CH2

O

S

NH2

C

3HC

O Ni

X

Ni

N

N

N

H2N

N

O

OX

Y

NH2

CH

CH2

O

N

NH

NCH

CH2

O

N

NH

NH

CHCH2

O

N

NH

NH

CH

CH2

O

N

NH NH

CH

CH2

O

OH

OH

NiNH

CHCH2

O

S

H3C

N

CH

CH3

O

NCH

CH2

O

N+

NH

OH

Ni

X

NH2CH

CH2

O

S

N

CHCH2

O

S

NH2

Ni

Witkowska D., Politano R., Rowińska-Żyrek M., Guerrini R., Remelli M., Kozłowski H., Chem. Eur. J., 2012, 18, 11088-11099.

Binding modes of nickel to various Hpn sequences

MAHHEEQHGG HHHHHHHTHH HHYHGGEHHH

HHHSSHHEEG CCSTSDSHHQ EEGCCHGHHE

Competition plots of Ac-THHHHHYHGG-NH2 and MAHHEEQHG-NH2 with Cu2+ and Ni2+

His-rich complexes are more stable only at below–physiological pH

Rowińska-Żyrek M., Witkowska D., Potocki S., Remelli M., Kozłowski H., New J. Chem., 2013, 37, 58-70.

Ni

N

N

N

H2N

N

O

OX

Y

ALBUMIN-LIKE BINDING

Xaa-Yaa-His-

MAHHEQQQQQQA-NH2 (Hpn-like) MAHHEEQHG-NH2 (Hpn) Ac-EEGCCHGHHE-NH2 (Hpn) Ac-THHHHYHGG-NH2 (Hpn) Ac-ACCHDHKKH-NH2 (HspA)

Copper and nickel binding to fragments

of Helicobacter pylori proteins

4 6 8 10

0

20

40

60

80

100

free Ni2+

Ni2+

-MAHHEEQHG-NH2

Ni2+

-MAHHEQQQQQQA-NH2

% f

orm

ation r

ela

tive t

o N

i2+

pH

D. Witkowska, S.Bielińska, W.Kamysz and H.Kozlowski, J. Inorg. Biochem., 2011, 105, 2, 208-214

The distribution of Ni2+ between MAHHEEQHG-NH2

and MAHHEQQQQQQA-NH2

(0.5:1:1) The N-terminal fragment of Hpn-like (MAHHEQQQQQQA-NH2) is much more effective in binding both Cu2+ and Ni2+ ions than the N-terminal fragment of Hpn

MAHHE-NH2...M2+

MAHHEEQHG-NH2...M2+

MAHHEQHQA-NH2...M2+

MAHHEQQQQQQA-NH2...M2+

Cu2+

MAHHEQQQQQQA-NH2...Cu2+

Common M2+ binding

pattern

Ni2+

MAHHEEQHG-NH2...Ni2+

Method: full DFT

(M052X/6-31G(d,p)

Full optimization of the

structure of the complex

(M=Cu, Ni)

N.M. Chiera, M. Rowinska-Zyrek, R. Wieczorek, M. Remelli, H. Kozlowski, Metallomics, 5, 214-221, (2013)

MAHHEEQHG-NH2...Ni2+

Ni2+

MAHHEQQQQQQA-NH2...Ni2+

Ni2+

Method: DFT (M052X/6-31G(d,p) Gln-rich

fragment influence on peptide folding

N.M. Chiera, M. Rowinska-Zyrek, R. Wieczorek, M. Remelli, H. Kozlowski, Metallomics, 5, 214-221, (2013)

CONCLUSIONS

Cys-Xaa-Yaa----Cys sites are much more efficient binding sites for Zn(II) when compared to Ni(II)

Poly-Gln sequences may stabilize the metal ion binding to peptide by organizing specific structure around the binding site with hydrogen bond network of side-chain amide groups.

Cys-Cys is a basic and very specific binding site for Ni(II) ions as that of albumin but much more efficient

His-rich complexes are more stable only at below–physiological pH

The thermodynamic stability of Bi(III) complexes is several orders of magnitude higher than those of Ni(II). Bi(III) is able to displace Ni(II) from its binding sites.

Overall binding process of hexahistidine to Ni2+-NTA at moderate concentration. Ni2+

is immobilized to the surface of the sensor chip via NTA. One imidazole group of the hexahis-tag (a) makes first contact with Ni2+ (b). After the monovalent interaction is established the high local concentratiom of his ligand facilitates the interaction with a second imidazole forming a divalent complex with histidine from the i and i+2 position (c). By consecutive dissociation and reassociation (d, e) the molecule ‘slides’ over the chip surface, which explains the observed rebinding effect in the SPR measurments. Finally, the hexahis dissociates from the Ni2+ (f, g).

S. Knecht, D. Ricklin, A. N. Eberle and B. Ernst, J. Mol. Recognit., 2009, 22(4), 270-279.

The image of the His-tag motifs after energy minimization calculations. (a) His-tag2; (b) His-tag3; (c) His-tag4; (d) His-tag5; and (e) His-tag6. Hsuan-Liang Liu, Yih Ho, Chia-Ming Hsu, J. Biomol. Struct. Dynam., 2003, 21(1), 31-40.

Important issues:

There is a relationship between the number of residues in the His-tag and their properties of binding

A bivalent ligand formed by two His at positions n+2 and n and n+5 in hexapeptide are better than the other possible divalent ligands (n+1, n+3, n+4)

The elongation His-tag for example to seven or ten histidines in not profitable to metal ion binding.

Binding affinity KA (1/KD) of different oligohistidines to Ni2+-NTA chips in surface

plasmon resonance experiment.

S. Knecht, D. Ricklin, A. N. Eberle and B. Ernst, J. Mol. Recognit., 2009, 22(4), 270-279

Ac-HHHHHH-NH2 – His-Tag uses in ITC

Ac-EDDHHHHHHHHHG-NH2 – sequence

occurs in nature (from venom glands of the snake)

Ac-HHHHHH-NH2 – log b: 11,61

Ac-EDDHHHHHHHHHG-NH2 – log b: 16,82

We do not know why ???

2 3 4 5 6 7 8 9 10 11

0

20

40

60

80

100

Cu2+

- Ac-HHHHHH-NH2

free Cu2+

Cu2+

- Ac-EDDHHHHHHHHHG-NH2

% fo

rma

tion

re

lativ

e to

[C

u2

+]

pH

2 3 4 5 6 7 8 9 10 11 12

0

20

40

60

80

100free Cu

2+

Cu2+

- Ac-THHHHAHGG-NH2

Cu2+

- Ac-HHHHHH-NH2

% fo

rma

tion

re

lativ

e to

[C

u2

+]

pH

2 3 4 5 6 7 8 9 10 11 12

0

20

40

60

80

100free Cu

2+

Cu2+

- Ac-PVHTGHMGHIGHTGHTGHTGSSGHG-NH2

Cu2+

- Ac-HHHHHH-NH2

% fo

rma

tio

n r

ela

tive

to

[C

u2

+]

pH

2 3 4 5 6 7 8 9 10 11

0

20

40

60

80

100

free Cu2+

Cu2+

- EDDHHHHHHHHHGVGGGGGGGGGG-NH2

Cu2+

- Ac-EDDHHHHHHHHHG-NH2

% fo

rma

tio

n r

ela

tive to

[C

u2

+]

pH

We do not know exactly which histidine residues are bound.

WHY POLY-HIS SEQUENCE IS SO EFFECTIVE IN METAL ION BINDING BINDING?

IT IS VERY A GOOD QUESTION

He

Li Be B Ne

Al Ar

Sc Ti Cr Ga Ge As Br Kr

Rb Sr Y Zr Nb Ru Rh Pd Ag Cd In Sb Te Xe

Cs Ba La Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi

La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Lu

C

Pt Au

Cr V

Ca Se

Li

99mTc

Fe Mg

Cu

Gd

I

F

Zn

Ag

Al

67Ga

B

N2O

N O

S

Ba

Na

K

A Periodic Table of Medicines

P

Sb

Bi

Mn

Mo

Co Si

Sr Sn

Ti

133Xe 201Tl

As 90Y

188Re 153Sm

The control of urease and hydrogenase synthesis in H. pylori

Ni

Ni

Ni Ni

NixA

HspA Hpn

HypA HypB UreH

UreG

UreF

UreE

UreI

UreA UreB

Ni

Ni

Ni

Ni

Ni

Ni

Ni

ApoNikR

Ni

urease

Ni

+Ni

Ni

Ni

NixA

to urease or

hydrogenase

Ni

Ni

activation of

nik operon

Levels of control: •Expression of UreA/B subunits controled by acidity and nickel-responsive transcriptional regulator NikR •Incorporation of Ni(II) into urease mediated by UreEFGH and HypA/B accessory proteins • Ni(II) availability in the cytoplasm influenced by the NixA importer and the amount of His-rich proteins such as Hpn and HspA •Urea substrate entry via UreI

4 6 8 10

0

20

40

60

80

100%

fo

rma

tio

n r

ela

tive

to

[C

u2

+]

pH

free Cu2+

DDD

ADSG

ADSGEGDFLAEGGGVR

EDDHHHHHHHHHGVGGGGGGGGGG-NH2