Analyses of structural rearrangements of membrane proteins by fluorescent unnatural amino acid scanning

2016 Fiscal Year Final Research Report

• Project/Area Number: 15K15036
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: General physiology
• Research Institution: National Institute for Physiological Sciences
• Principal Investigator: KUBO Yoshihiro 生理学研究所, 分子細胞生理研究領域, 教授 (80211887)
• Research Collaborator: Tsari Andriani Rizki 生理学研究所, 分子細胞生理研究領域, 大学院生
• Project Period (FY): 2015-04-01 – 2017-03-31
• Keywords: 蛍光非天然アミノ酸 / P2X2 / ATP受容体チャネル / 動的構造変化

Outline of Final Research Achievements

An experimental method has attracted attention which approaches structural rearrangement of membrane proteins by detecting the fluorescent intensity change of fluorescent unnatural amino acid incorporated at the position of stop codon introduced by mutation. We first optimized its various experimental conditions and then applied it to the ATP and voltage gated structural rearrangements of ATP receptor channel P2X2. We observed the followings. (1) ATP evoked structural rearrangements occur globally at various parts of P2X2. (2) At Ser54 in the transmembrane region 1, the fluorescent intensity change was much slower than the current activation upon ATP application, suggesting secondary structural rearrangements such as pore dilation. (3) Fluorescent change associated with voltage dependent gating was observed at T339 in the transmembrane region 2.

Free Research Field

分子生理学