NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase
Source: The Journal of Biochemistry, Volume 151, Number 3, 13 March 2012 , pp. 335-342(8)
Publisher: Oxford University Press
Abstract: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 1H-15N HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the -barrel at 45C, which is around the Tm value for the N I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54C, where the maximum accumulation of the intermediate state was found. 1H-NMR and CD measurements showed that the Tm values obtained by monitoring Trp54 at the upper region and Trp43 at the bottom region of the -barrel were 41.4 and 47.5C, 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.
Document Type: Regular paper
Affiliations: 1: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531; , Research Fellow of the Japan Society for the Promotion of Science, 8 Ichiban-cho, Chiyoda-ku, Tokyo 102-8472, , 2: School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337; , 3: Department of Food and Nutrition, Tsu City College, 157 Ishinden-Nakano, Tsu, Mie 514-0112; and , 4: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531; , 5: Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan,
Publication date: 2012-03-13