Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

ANBF, 04/05 AB06

X-ray Absorption Studies of Metal Binding to the Amyloid β-peptide
1

Victor A. Streltsov*1, Garry J. Foran3, Kevin J. Barnham2 and Jose N. Varghese1
CSIRO Health Sciences & Nutrition, 343 Royal Parade, Parkville, Vic 3052, Australia
2
University of Melbourne, Vic 3010, Australia
3
KEK-PF, Tsukuba, Ibaraki 305-0801, Japan

Introduction
Alzheimer’sdisease(AD)
isprogressive
neurodegenerative disorder that is characterised by thepresence of misfolded protein deposition, described asamyloid plaque. This structural transition from the native
state to a β-sheet aggregated form is accompanied by again of toxic function. The major constituent of AD
plaques is the 4.3kDa amyloid beta peptide (Aβ) that iscleaved from the membrane-bound amyloid precursor
protein (APP). In vitro, Aβ binds metal ions including
2+2+3+Cu , Zn , and Fe giving rise to peptide aggregation andextensive redox chemical reactions. Since elevated levelsof these metals are found in amyloid deposits in ADaffected brains, the oxidative stress causing cellulardamage may be related to the production of reactive
oxygen species by metallated forms of Aβ [1,2].
NMR and EPR results [2] indicate the coordination
2+2+sphere about the Cu and Zn ions may be similar to thatobserved in superoxide dismutase (SOD) with His6,His13 and His14 involved. The presence of bridging
histidine between two metal ions, as found in the active
site of SOD, was inferred.
The studies carried out so far provide an insight into
the nature of the metal binding to Aβ but this is an
incomplete view. Our recent XAFS studies onAβ−metal complexes
havebeenfocusedincharacterization of Cu(II) and Zn(II) binding to the Aβpeptide under a range of conditions varying metal/peptide
concentrations, pH, and duffers.
Experimental
Due to limited solubility of the Aβ itself in the presenceof metals in an aqueous environment the shortened Aβ(116) and Aβ(1-28) forms have also been used inexperiments. Shorter forms contain all the residues formetal binding and are soluble at millimolar
concentrations with Cu or Zn bound. EPR suggests thatmetal binding is the same for the shorter peptide.
2+Samples with equimolar Aβ(1-42) and Zn , Aβ(1-28) and2+2+2+(Zn + Cu ) in PBS, pH 6.4; Ab(1-28) and Cu in PBS,
pH 7.4, and in Ethyl Morpholine, pH 7.0, weresynthesized as described previously [2]. There is enoughevidence in the literature to suggest that there are pH
driven changes in the metal coordination sphere. There
are also buffer driven differences in the coordination
sphere; although these are likely to be more obvious withthe longer peptides with greater aggregation tendencies
The samples were transported and kept in liquid
nitrogen prior the experiments. The solution volumes of0.1-0.2mL were injected into specially designed solution
cells [4] made of Teflon with the Kapton tape sealed
windows matching the size of the beam. The solution

cells were inserted into a Crydone REF-1577-D22 closedcycle cryostat and were maintained at ~10K with a
Neocera LTC-11 temperature controller unit.
The Cu K-edge XAS was measured at the Australian
National Beamline Facility (ANBF) on bending magnet
beamline 20B using a Si(111) monochromator. XAFS
data analysis was performed using the XFIT package [5].
The scans were averaged using weights based on
mismatch between the XAFS and the average XAFS. A
background correction was applied by fitting spline
curves. The background-subtracted, normalized data were3
converted to k space, where k is the photoelectron wave
vector, to enhance the amplitude of the high k oscillations.
Results and Discussion
Physically sensible structural models have beenobtained using restrained refinements (R-factor range of2+sample
20-25%). Example of the Aβ(1-42)+Zn
refinement is shown below .

Preliminary structural models obtained from EXAFS
data are consistent with recent independent EPR
measurements and models proposed in [3]. Further
accurate measurements will be required to check
reproducibility of results obtained.
References[1] A. I. Bush, TINS, 26, 207 (2003).
[2] C. C. Curtain et al., JBC, 276, 20466 (2001).
[3] C. D. Syme et al., JBC, 279, 18169 (2004).
[4] S. Best, Private Communication.
[5] P. J. Ellis et al., J. Synch. Rad. 2, 190 (1995).
* victor.streltsov@csiro.au

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