Authors: Miyamoto, Yuya¹; Noda, Yasuo²; Iida, Tsukimi³; Yamaguchi, Keisuke⁴; Nishimura, Shigenori⁴; Tanaka, Akiyoshi⁵; Segawa, Shin-ichi²; Inui, Takashi⁴

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 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 54C, where the maximum accumulation of the intermediate state was found. ¹H-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 -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.

Keywords: conformational change; intermediate state; lipocalin-type prostaglandin D synthase; NMR spectroscopy; thermal unfolding

Document Type: Regular paper

DOI: http://dx.doi.org/10.1093/jb/mvr140

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

Related content

• In this: publication
• By this: publisher
• By this author: Miyamoto, Yuya ;  Noda, Yasuo ;  Iida, Tsukimi ;  Yamaguchi, Keisuke ;  Nishimura, Shigenori ;  Tanaka, Akiyoshi ;  Segawa, Shin-ichi ;  Inui, Takashi

Website © Publishing Technology. Article copyright remains with the publisher, society or author(s) as specified within the article.

• Terms and conditions
• Privacy policy
• Information for advertisers