CD spectra of 20 µM PrP(111-126) with varying Cu²⁺ concentrations (0, 0.3, 0.6, 0.9, 1.5, 1.8 mol. equiv) in PBS at 20 °C.

<p>(A) UV-CD and (B) visible-CD spectra of PrP(111-126) with 0 to 1.8 mole equivalent of Cu²⁺. (B, inset) intensity of 560 nm CD band vs. Cu²⁺ concentration. (C) CD spectra of PrP(111-126) with or without sonication for 120 seconds in the presence of 1 mole equivalent Cu²⁺.</p

Solvent Microenvironments and Copper Binding Alters the Conformation and Toxicity of a Prion Fragment

<div><p>The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu²⁺ ions inhibited the structural transition and fibril formation of the peptide in a cell free <i>in vitro</i> system. The fact that Cu²⁺ supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu²⁺ ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu²⁺ ions, owing to binding affinity of copper towards histidine moiety present in the peptide. </p> </div

Morphology of fibrillar assemblies of the PrP

<p>(111-126). AFM height images of (A) PrP(111-126) and (B) PrP(111-126) in the presence of Cu²⁺ ions. Height profiles of the fibrils for lines marked on the images: (C) PrP (111-126) and (D) PrP (111-126) with Cu²⁺. Scale bar represents 500nm.</p

Cell viability (MTT) assay.

<p>Monomeric and fibrillar form of PrP(111-126) and PrP(113-127) were treated to astrocyte cultures. (A) Cytotoxicity of monomers and fibrils at various concentrations of PrP(111-126) and at 25µM of PrP(113-127). (B) PrP(111-126) (20µM) in the presence of different concentrations of Cu²⁺and 25µM of PrP(113-127) with 25µM Cu²⁺.</p

Effect of microenvironment on the secondary structure of PrP

<p>(111-126). (A) CD spectra of 20µM PrP(111-126) in different TFE concentrations, 0% to 100% TFE, in PBS at 4 °C. (B) CD spectra of PrP(111-126) at pH 2, pH 5, pH 7, and pH 10.6 at 20°C. (C) CD spectrum of PrP(111-126) in 0.1%(CMC) of SDS, and in liposomes in deionized water at 20 °C. (D) surface area-pressure isotherm of PrP(111-126) at air-water interface, and (inset) CD spectrum of peptide film (20 layers) on quartz showing β-sheet formation.</p