Abstract

The conformational change of the recombinant, murine prion protein (PrP) from an α-helical to a β-sheet enriched state was monitored by time-resolved Fourier transform infrared (FT-IR) spectroscopy. The α-to-β transition is induced by reduction of the single disulfide bond in PrP. This transition is believed to generate the scrapie form PrPSc, the supposed infectious agent of transmissible spongiform encephalopathies. We followed the kinetics of this conformational change using a novel method for amide I band analysis of the infrared (IR) spectra. The amide I analysis provides the secondary structure. The amide I decomposition was calibrated with the three dimensional structure of cellular PrP solved by nuclear magnetic resonance (NMR). The novel secondary structure analysis provides a root mean squared deviation (RMSD) of only 3% as compared to the NMR structure. Reduction of α-helical PrP caused the transient accumulation of a partially unfolded intermediate, followed by formation of a state with higher β-sheet than α-helical structure contents. The novel approach allows us to now determine the secondary structure of the β-sheet conformation. This was not determined by either NMR or X-ray. The experiments were performed in a double-sealed security cuvette developed for IR analysis of potentially infectious PrP samples outside the biosafety laboratory.

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