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Spectroscopic Studies of Copper Binding to Methionine and Histidine-Rich hCtr1 Model Peptides. Kathryn L. Haas Department of Chemistry Duke University April,4 2006. Neurological function (dopamine β hydroxylase). Copper in Human Health. Oxidative phosphorylation (cytochrome C-oxidase).

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Spectroscopic Studies of Copper Binding to Methionine and Histidine-Rich hCtr1 Model Peptides


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spectroscopic studies of copper binding to methionine and histidine rich hctr1 model peptides

Spectroscopic Studies of Copper Binding to Methionine and Histidine-Rich hCtr1 Model Peptides

Kathryn L. Haas

Department of Chemistry

Duke University

April,4 2006

copper in human health

Neurological function(dopamine β hydroxylase)

Copper in Human Health

Oxidative phosphorylation(cytochrome C-oxidase)

  • Important for redox chemistry

Cu(II) + e‾ Cu(I)

  • Unregulated redox is dangerous

Antioxidant activity (Cu/Zn superoxide dismutase)

Iron metabolism(ceruloplasmin)

Fenton Chemistry

Cu+ + H2O2 Cu2+ + HO‾ + HO•

Oxidative Stress!

Pigmentation(tyrosinase)

Connective tissue formation(lysyl oxidase)

2

Waggoner, Neurobiol. of Disease, 1999, 6, 221

copper in human disease

Neurological function(dopamine β hydroxylase)

Copper in Human Disease

Oxidative phosphorylation(cytochrome C-oxidase)

  • Amyotrophic Lateral Sclerosis (ALS)1
    • SOD1 mutation enhances free radical generation by Cu
  • Alzheimer’s Disease2
    • Cu may promote Aβ aggregation
  • Prion Disease3
    • Cu-binding to prion protein enhances protease stability

1. Rasia, PNAS, 2005, 102(12), 4294.

2. Bush, PNAS, 2003, 100(20), 11193.

3. Sigurdsson, J. Biol. Chem., 2003, 278(47), 46199

Antioxidant activity (Cu/Zn superoxide dismutase)

Iron metabolism(ceruloplasmin)

Pigmentation(tyrosinase)

Connective tissue formation(lysyl oxidase)

3

Waggoner, Neurobiol. of Disease, 1999, 6, 221.

menke s and wilson s disease
Menke’s and Wilson’s Disease

Lutsenko, S. et. al., J. Membrane Biol., 2002, 191, 1.

MNKP and WNDP are P-type ATPase polytopic membrane proteins and have 55% amino acid identity

ctr copper transporter required for extracellular copper acquisition
Ctr: Copper Transporter Required for Extracellular Copper Acquisition

O’Halloran, J. Bio. Chem., 2000, 275(33), 25057.

architecture of the ctr copper transporter
Architecture of the Ctr Copper Transporter

Aller, PNAS, 2006, 103(10), 3627.

how do cells regulate cu uptake
How do cells regulate Cu-uptake?
  • Copper transport is passive
    • ATP synthesis inhibitors have no effect on Cu uptake
    • Na+/K+-ATPase inhibitors have no effect on Cu uptake
  • Copper must always be bound to proteins to prevent toxicity
    • Therefore transport must be governed by exchange of copper ions with delivery proteins, chaperones, and small chelators

Binding site affinity and structure isimportant for control

hctr1 human high affinity copper transporter
hCtr1: Human High Affinity Copper Transporter

Mets motif = MXnMXmM n,m=1 or 2

Glycosylation on N15

Cu chaperone

Delivery of Cu(I) to appropriate cuproenzyme

n terminal hctr1 model peptides
N-Terminal hCtr1 Model Peptides

By standard F-moc solid phase peptide synthesis

esi ms hctr1 14
ESI-MS (+)hCtr1-14

P++/2

hCtr1-14

P+

H2N M D H S H HM G M S Y M D S

+ CuSO4

P++/2

[PCu(II)]++/2

PCu(II)+

P+

[Cu]

P++/2

+ CuSO4 + H2Asc

[PCu(I)]++/2

P+

PCu(I)+

band typical of His-Cu(II) bindingTitration of 400μM hCtr1-14 with 0-600 μM CuSO4

13

esi ms hctr7 14k

hCtr7-14K

P+

ESI-MS (+)hCtr7-14K

Ac M G M S Y M D S K

Mets motif MXMXXM is capable of binding Cu and is selective for Cu(I)

P+

+ CuSO4

+ CuSO4 + H2Asc

PCu(I)+

[PCu(I)]++/2

P+

esi ms hctr38 45k

hCtr38-45K

P+

ESI-MS (+)hCtr38-45K

Ac S M M M M P M T K

Mets motif MMMMXM is capable of binding Cu and is selective for Cu(I)

P+

+ CuSO4

P+

+ CuSO4 +H2Asc

[PCu(I)]++/2

16

determination of k d by peptide inhibition of copper catalyzed ascorbate oxidation

Cu chelation slows rate

Determination of KD by Peptide Inhibition of Copper-Catalyzed Ascorbate Oxidation

Rate limiting step

HAsc‾ HAsc• Asc

determination of k d by peptide inhibition of copper catalyzed ascorbate oxidation20

Determination of KD by Peptide Inhibition of Copper-Catalyzed Ascorbate Oxidation

HAsc‾ HAsc• Asc λmax = 260nm no absorbance at 260nm

slide21

Pseudo 1st Order Kinetics

-d[HAsc-]/dt = k[HAsc-][Cu2+]

Under excess HAsc-

kobs = k[Cu2+]

-d[HAsc-]/dt = kobs[HAsc-]

current understanding
Current Understanding
  • MXmMXnM motifs are sufficient for binding Cu(I) with a KD of ~3-6μM
  • His cluster HHXH contributes to Cu(II) binding with a KD ~ 1μM
  • Further effort needs to be taken to understand effect of His residues on Cu(I) and Cu(II) binding
n terminal hctr1

7-14

1-14

38-45

MDHSHH MGMSYMDS NSTMQPSHHHPTTSASHSHGGGDS SMMMMPMT FYFGFKNVELLFSGLVINT

N-Terminal hCtr1
  • Current studies are limited because isolated sequences may not indicate binding of overall N-terminal hCtr1
expression of 65aa n terminal in e coli
Expression of 65aa N-Terminal in E.coli

Ampr

Obtained from Thiele Lab

expression of 65aa n terminal in e coli25

Ampr

Expression of 65aa N-Terminal in E.coli

Competent E. coli

Purified N-hCtr1

XarrestAffinity purification

Expression of GST-N-hCtr1

Solution of GST+Factor Xa + N-hCtr1

Factor Xa

GST Affinity purification

IsolatedGST-N-hCtr1

GST Affinity purification

so far

1 2 3 4 5 6 7 8 9 10

GST-N-hCtr1

“N-hCtr1”7234Da

So Far…

1 blank

2 Crude induced lysate

3 Buffer

4 Purified fusion protein

5 Factor Xa cleavage RXT

6 Factor Xa

7 GST affinity purification

8 Xarrest affinity purification

9 Both affinity purifications

10 SDS-PAGE broad range standard

37Kda

7Kda

future studies on n terminal hctr1
Future Studies on N-Terminal hCtr1

Observe overall structural changes upon Cu binding using Circular Dichroism (CD) and 15N NMR

Wawick Analytical Service. Available at http://www.warwickanalytical.co.uk/circular.htm