title: Structural characteristic of folding/unfolding intermediate of pokeweed anti-viral protein revealed by time-resolved fluorescence.
abstract: The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.
authors: Industrial Technology Center of Nagasaki, Ohmura, Nagasaki, Japan.
authors: Shuzo
authors: S
authors: Matsumoto
authors: Yuka
authors: Y
authors: Taniguchi
authors: Yukihiro
authors: Y
authors: Fukunaga
authors: Hiromichi
authors: H
authors: Nakashima
authors: Keiichi
authors: K
authors: Watanabe
authors: Shoji
authors: S
authors: Yamashita
authors: Etsuko
authors: E
authors: Nishimoto
mesh_terms: D004735:Energy Transfer; D005454:Fluorescence Polarization; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D054789:Ribosome Inactivating Proteins, Type 1 / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D054789:Ribosome Inactivating Proteins, Type 1; C024275:pokeweed antiviral protein