Structural analysis of Am-resistant M2-S31N mutant using a novel detergent and solution NMR

2015 Fiscal Year Final Research Report

• Project/Area Number: 26893117
• Research Category: Grant-in-Aid for Research Activity Start-up
• Allocation Type: Single-year Grants
• Research Field: Physical pharmacy
• Research Institution: Kyoto University
• Principal Investigator: Kawano Kenichi 京都大学, 物質-細胞統合システム拠点, 研究員 (70732874)
• Project Period (FY): 2014-08-29 – 2016-03-31
• Keywords: 可溶化剤 / M2プロトンチャネル / S31N変異体 / 溶液NMR / A型インフルエンザウイルス / アマンタジン耐性 / 二量体

Outline of Final Research Achievements

The synthesis and purification methods of a novel detergent were established. Before applying it to M2 protein, the well-studied photosensitive bacteriorhodopsin (bR) was used as a model membrane protein to examine the solubilization ability of the detergent, and the time-dependent thermal stability of bR in the detergent micelle, monitoring change in the absorbance spectrum. As a result, the abilities of the detergent were proven to be significantly improved compared with the existing detergents. The micelle size after solubilizing bR was available for solution NMR. Although the expression of M2 protein was confirmed by the Western blotting, only a part of M2 protein formed the dimers crosslinked by the disulfide bonds at the N-termini. To facilitate the crosslinking, a self-associating coiled-coil sequence was inserted into the N-terminus of the M2 protein. The structural analysis of the dimeric M2 protein will be started following confirmation of M2 dimerization.

Free Research Field

物理化学