演題概要

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第3日 5月19日(金) 9:50~10:10 C会場(101)

天然変性蛋白質Mint3とFIH-1の相互作用解析におけるH/D交換質量分析法の適用

(1東大院工2東大院新領域3東大医科研4金沢大医薬)
o展天承1中山佳昭2長門石曉3坂本毅治3清木元治4津本浩平1,2,3

In this research, we combined a series of physicochemical analysis with H/D exchange mass spectrometry (HDX-MS) to elucidate the binding mechanism of Mint3 to FIH-1.
First of all, we identified the N-terminal fragment of Mint3 (Mint3NT), the region that is capable of binding to FIH-1, as an IDP by circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and HDX-MS. Then, we performed isothermal titration calorimetry (ITC) analysis to characterize the interaction. The result indicates that Mint3NT has at least two core regions for binding to FIH-1, and FIH-1 needs α-keto glutaric acid (α-KG) and Fe2+ as cofactors for acquiring high affinity with Mint3NT. It is also suggested that the conformation of Mint3NT is fixed through the interaction, since -T∆S was very large. However, it is still unclear whether a secondary-structure is formed or not in the complex. Finally, we performed HDX-MS analysis to determine the binding sites of both proteins. Consequently, the region of FIH-1, which is identified to be affected by binding of Mint3NT, is consistent with the region affected by α-KG. Thus, we conclude that the binding sites of FIH-1 are located in the both ends of FIH-1 dimer.