2 research outputs found
Synthesis of double-fluorescent labeled prion protein for FRET analysis
<div><p>An abnormal form of prion protein (PrP) is considered to be the pathogen in prion diseases. However, the structural details of this abnormal form are not known. To characterize the non-native structure of PrP, we synthesized position-specific double-fluorescent labeled PrP for a fluorescence resonance energy transfer (FRET) experiment. Using FRET, we observed a conformational change in the labeled PrP associated with amyloid fibril formation. The FRET analysis indicated that the distance between fluorescent labeled N- and C-terminal sites of PrP increased upon the formation of amyloid fibrils compared with that of the native state. This approach using FRET analysis is useful for elucidating the structure of abnormal PrP.</p></div
Nearly Reversible Conformational Change of Amyloid Fibrils as Revealed by pH-Jump Experiments
pH-jump
induced conformational transitions between substates of
preformed amyloid fibrils made by a fragmented peptide of helix 2
(H2 peptide) of MoPrP were detected, and their kinetics were analyzed
using a novel pH-jump apparatus specially designed for observing amyloids.
Previously, we reported that H2 peptide formed ordered fibrils with
a minimum at 207 nm on CD spectra at pH 2.9 (named pH 2.9 fibrils),
but formed aggregate-like fibrils with a minimum at 220 nm at pH 7.5
(named pH 7.5 fibrils). When pH-jump from 2.9 to 7.5 was performed,
the CD spectrum changed instantly, but the finally observed ellipticities
were clearly distinct from those of pH 7.5 fibrils. Thus, the finally
observed state is termed ‘pH 7.5-like fibrils’. However,
pH 7.5-like fibrils reverted to the conformation very similar to that
of the pH 2.9 fibrils when the pH of the solution was restored to
2.9. Then, we examined the kinetics of the nearly reversible conformational
changes between pH 2.9 fibrils and pH 7.5-like fibrils using ANS fluorescence
stopped-flow, and we observed relatively fast phases (0.7–18
s<sup>–1</sup>). In contrast, the conversion between pH 7.5-like
fibrils and pH 7.5 fibrils never occurred (<0.2 day<sup>–1</sup>). Thus, H2 fibrils can be switched readily between distinct conformations
separated by a low energy barrier, while a large energy barrier clearly
separated the different conformations. These conformational varieties
of amyloid fibrils may explain the physical basis of the diversity
in prion