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Resonance Raman studies on functional mechanisms of light-driven proton pumps

塩谷, 智巳 大阪大学 DOI:10.18910/85329

2021.09.24

概要

【イントロダクション】
グロイオバクターロドプシン(GR)は、光駆動外向きプロトンポンプである。タンパク 質部分オプシンと、その内部にレチナール発色団を有する。GRでは、内部のレチナール発色団の光異性化に誘起されてプロトン移動が起きる。本研究では、Trp222残基をPheに置換したW222F変異体では、プロトン輸送活性が 9%に低下することを発見した。そこで、W222F変異体の時間分解共鳴ラマンスペクトルを測定し、発色団の構造変化と、オプシンとの相互作用について議論した。

【実験手法】
試料は、大腸菌に発現させたタンパク質を可溶化・精製し、調製した。紫外共鳴ラマン測定には、波長233 nmのプローブ光を用い、300 μs-10 msの範囲で時間分解スペクトル得た。可視共鳴ラマン測定には、波長475 nmのプローブ光を用い、50 ns-100 μsの範囲で時間分解スペクトルを得た。どちらも波長532 nmのポンプ光を用いた。

【結果・考察】
Figure 1に、野生型(WT)とW222F変異体(B)の時間分解紫外共鳴ラマンスペクトルを示す。WTでは、 Trp残基とTyr残基由来のラマンバンドに大きな変化があり、プロトン輸送過程にタンパク質が大きく動くことが明らかになった。一方でW222F変異体では、WTでみられたスペクトル変化が観測されなかった。この結果 Figure 1. UV resonance Raman spectra of GR-WT (A) and the W222F mutant (B). は、W222F変異体ではタンパク質部分が構造変化しなかったこと示唆した。

Figure 2に、未反応状態および光励起後100 μsにおけるC-C伸縮振動バンド領域の可視共鳴ラマンスペクトルを示す。WTと変異体とともに、未反応状態柄では~1200 cm-1、100 μsでは1185 cm-1にバンドが観測された。前者はall-trans形、後者は 13-cis形を示すマーカーバンドである。この結果から、変異体はWTと同様にall-trans形から13-cis形へ光異性化を起こすことが明らかになった。

W222F変異体において、発色団がWTと同様な構造変化をするにもかかわらずプロトン輸送活性が大きく低 下するという事実は、発色団の構造変化がTrp222残基との接触を介してオプシンと相互作用し、プロトン輸 送を誘起することを示唆している。

論文中には、光駆動内向きプロトン輸送タンパク質のレチナール発色団構造を調べた成果も報告している。

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