Copper(II) Binding Modes in the Prion Octapeptide PHGGGWGQ: A Spectroscopic and Voltammetric Study

Abstract

The N-terminal octapeptide repeat region of human prion protein (PrPc) is known to bind Cu(II). To investigate the binding modes of copper in PrPc, an octapeptide Ac-PHGGGWGQ-NH2 (1), which corresponds to an octa-repeat sequence, and a tetrapeptide Ac-HGGG-NH2 (2) have been synthesised. The copper(II) complexes formed with 1 and 2 have been studied by circular dichroism (CD) and electron spin resonance (ESR) spectroscopy. Both peptides form 1:1 complexes with Cu(II) at neutral and basic pH. CD, ESR and visible absorption spectra suggest a similar co-ordination sphere of the metal ion in both peptides, which at neutral pH consists of a square pyramidal geometry with three peptidic nitrogens and the imidazole nitrogen as donor atoms. Cyclic voltammetric measurements were used to confirm the geometrical features of these copper(II) complexes: the observation of negative redox potentials are in good agreement with the inferred geometry. All these results taken together suggest that peptide 1 provides a single metal binding site to which copper(II) binds strongly at neutral and basic pH and that the binding of the metal induces the formation of a stiffened structure in the HGGG peptide fragment.

Copper and the Prion Protein: Methods, Structures, Function, and Disease

“…First, the variable structures within the octarepeat domain (Figure 8), which depend on copper concentration, are certainly not characteristic of a well-defined metal-ion-coordination site typically required for catalysis. Moreover, even the highest affinity determined from our measurements is significantly less than that found for copper-dependent SODs, which bind with K d values on the order of 10 −14 M. Finally, the strong ligand field provided by the negatively charged amide nitrogens, in the dominant component 1-coordination mode, preferentially stabilizes the Cu 2+ -oxidation state, and direct electrochemical measurements have confirmed this (75).…”

Metal-Binding Ability of Human Prion Protein Fragment Peptides Analyzed by Column Switch HPLC

“…24) The interaction of full-length and truncated forms of PrP with Cu 2+ has been investigated using a range of techniques, including electron paramagnetic resonance (EPR), [25][26][27] We developed a column switch (CS)-HPLC system that can detect direct metal-binding to the octarepeat region of hPrP C , and the data obtained using our CS-HPLC method agreed well with previous CD analyses. 42,43) In this study, we used CS-HPLC to analyze the metal-binding characteristics of 21 synthetic fragment peptides derived from the sequence of hPrP amino acids 60-230.…”

“…[13][14][15][16][17] Although PrP metal-binding sites have been investigated using full-length PrP C or synthetic fragment peptides and it is now generally accepted that PrP C binds copper in vivo, 18) most researchers have focused on the octarepeat region, [19][20][21][22][23] and there are few reports describing the metal-binding ability of the middle-and C-terminal domains of hPrP. 24) The interaction of full-length and truncated forms of PrP with Cu 2+ has been investigated using a range of techniques, including electron paramagnetic resonance (EPR), [25][26][27] circular dichroism (CD), 28,29) X-ray crystallography 30) NMR, 31,32) MS, [33][34][35] Raman spectroscopy, 36,37) Fourier transform infrared (FT-IR) spectroscopy, 38) and potentiometry. 39) Although these methods have been useful, most can only detect metal-binding that occurs during conformational conversion, and as a result, they cannot detect metal-binding occurring without conformational conversion.…”

Prion Proteins with Insertion Mutations Have Altered N-terminal Conformation and Increased Ligand Binding Activity and Are More Susceptible to Oxidative Attack

“…1B. The three mAbs, 8B4 (residues [35][36][37][38][39][40][41][42][43][44][45], 8H4 (residues 175-185), and 8F9 (residues 220 -231), reacted equally with rPrP c and rPrP 8OR (Fig. 1C) (Fig.…”

“…The octapeptide repeat region of PrP c binds divalent cations such as Cu 2ϩ and Zn 2ϩ (39,40). However, whether such interactions are important in the pathogenesis of prion disease is not clear.…”