NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase

作者：Miyamoto Yuya; Noda Yasuo; Iida Tsukimi; Yamaguchi Keisuke; Nishimura Shigenori; Tanaka Akiyoshi; Segawa Shin ichi; Inui Takashi^*

来源：Journal of Biochemistry, 2012, 151(3): 335-342.

DOI：10.1093/jb/mvr140

• conformational change
• intermediate state
• lipocalin-type prostaglandin D synthase
• NMR spectroscopy
• thermal unfolding

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 H-1-N-15 HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the beta-barrel at 45 degrees C, which is around the T-m value for the N %26lt;-%26gt; I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54 degrees C, where the maximum accumulation of the intermediate state was found. H-1-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 beta-barrel were 41.4 and 47.5 degrees 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.

• 出版日期2012-3