NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase.
We previously reported that the thermal unfolding of mouse lipocalin-type prostaglandin D synthase (L-PGDS) is a completely reversible process under acidic conditions and follows a three-state pathway, including an intermediate state (I) between native state (N) and unfolded state. In the present study, we investigated the intermediate state of mouse C65A L-PGDS and clarified the local conformational changes in the upper and bottom regions by using NMR and CD spectroscopy. The (1)H-(15)N HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the β-barrel at 45°C, which is around the T(m) value for the N ↔ I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54°C, where the maximum accumulation of the intermediate state was found. (1)H-NMR and CD measurements showed that the T(m) values obtained by monitoring Trp54 at the upper region and Trp43 at the bottom region of the β-barrel were 41.4 and 47.5°C, respectively, suggesting that the conformational change in the upper region occurred at a lower temperature than that in the bottom region. These findings demonstrate that the backbone conformation of the bottom region is still maintained in the intermediate state.
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
Yuya
Y
Miyamoto
Yasuo
Y
Noda
Tsukimi
T
Iida
Keisuke
K
Yamaguchi
Shigenori
S
Nishimura
Akiyoshi
A
Tanaka
Shin-Ichi
S
Segawa
Takashi
T
Inui
D000818:Animals; D002942:Circular Dichroism / Q000379:methods*; D019746:Intramolecular Oxidoreductases / Q000737:chemistry*; D054834:Lipocalins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D051379:Mice; D011487:Protein Conformation; D011489:Protein Denaturation
D054834:Lipocalins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase