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Revealing Structural Changes of Prion Protein during Conversion from α‑Helical Monomer to β‑Oligomers by Means of ESR and Nanochannel Encapsulation
journal contribution
posted on 2015-02-20, 00:00 authored by Che Yang, Wei-Lin Lo, Yun-Hsuan Kuo, Jason
C. Sang, Chung-Yu Lee, Yun-Wei Chiang, Rita P.-Y. ChenUnder nondenaturing neutral pH conditions,
full-length mouse recombinant
prion protein lacking the only disulfide bridge can spontaneously
convert from an α-helical-dominant conformer (α-state)
to a β-sheet-rich conformer (β-state), which then associates
into β-oligomers, and the kinetics of this spontaneous conversion
depends on the properties of the buffer used. The molecular details
of this structural conversion have not been reported due to the difficulty
of exploring big protein aggregates. We introduced spin probes into
different structural segments (three helices and the loop between
strand 1 and helix 1), and employed a combined approach of ESR spectroscopy
and protein encapsulation in nanochannels to reveal local structural
changes during the α-to-β transition. Nanochannels provide
an environment in which prion protein molecules are isolated from
each other, but the α-to-β transition can still occur.
By measuring dipolar interactions between spin probes during the transition,
we showed that helix 1 and helix 3 retained their helicity, while
helix 2 unfolded to form an extended structure. Moreover, our pulsed
ESR results allowed clear discrimination between the intra- and intermolecular
distances between spin labeled residues in helix 2 in the β-oligomers,
making it possible to demonstrate that the unfolded helix 2 segment
lies at the association interface of the β-oligomers to form
cross-β structure.