Dynamics of sodium-driven stator units in bacterial flagellar motors

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题名:	Dynamics of sodium-driven stator units in bacterial flagellar motors
作者:	林再順;Lin, Tsai-Shun
贡献者:	物理學系
关键词:	細菌鞭毛馬達;bacterial flagellar motor;sodium-motive force;stator exchange;membrane protein;FliL
日期:	2021-10-25
上传时间:	2021-12-07 12:14:13 (UTC+8)
出版者:	國立中央大學
摘要:	許多細菌擁有奈米尺度的細菌鞭毛馬達結構，其大小約為50 奈米。透過轉動鞭毛馬達，細菌可以主動地在低雷諾數的環境中活動以取得維生的重要物質。一個鞭毛馬達的基本結構含有一條螺旋狀的鞭毛、一個轉子與為數約一打的定子單元。其中轉子是馬達的旋轉主體與鞭毛連接，帶動鞭毛轉動。而定子單元則是獨立的各自獨立的蛋白複合體，負責推動轉子轉動，其過程是藉由傳遞特定的離子(氫離子或鈉離子)至細胞體內，進而轉換離子驅動勢(Ion-movie force)至機械能。此外，定子單元的狀態在運轉的馬達周圍是動態的，有機率性的與馬達結合或分離。目前已知，與馬達結合的定子單元數目會隨環境浮動並且對於馬達的輸出至關重要。因此了解定子單元組裝的動力學特性對於解釋細菌鞭毛馬達的基本運作原理與特性相當的重要。這份論文的主要研究主要集中在鈉離子型態的定子單元上，其中包含了溶藻弧菌身上的原生定子，與人工合成表現在大腸桿菌上的嵌合體定子。我們應用了多種光學顯微技術來解析馬達上定子單元的數目與其動力學行為，應用的技術包含了全內反射營光顯微術 (TIRF)、光漂白螢光恢復術 (FRAP)、光啟動定位顯微術 (PALM)、與後焦面干涉術 (BFPI)。這些不同的技術，被整合到了一台商業化的通用顯微鏡上。除此之外，我們更進一步設計使該顯微系統加入了可以即時切換的注流系統，控制細菌的環境條件，用來做鈉離子濃度的切換；也加入了焦面鎖定系統，達到長時間觀測下穩定的影像。在這本論文中，主要探討三個與定子相關的計畫。第一個執行的計畫主要目標在於利用超解析顯微鏡PALM探討定子在馬達周圍的分布。根據實驗數據估計，在我們系統的PALM最佳的分辨率小於5奈米。當PALM應用在定位定子的實驗中，雙色螢光分子各別標記了轉子結構上的FliN蛋白與定子結構上的PomA蛋白。在經過仔細的量測，這兩個蛋白共定位的比例和化學固定對於實驗造成的影響後，我們發現定子在轉子周圍的密度，大約呈現定值而後隨距離增加而下降。然而，定子的密度分布卻遠低於現有模型的預測，根據該結果，我們提出未來在PALM實驗系統上優化的可能性；第二個計畫主要是分析溶藻弧菌中，馬達力矩輸出相關的FliL蛋白對於定子組裝於馬達上數量的影響。透過在TIRF顯微術下，計算馬達上FliL與定子的數量，其化學劑量在馬達上比值約為1:1。此外，當FliL蛋白被移除時，透過FRAP量測，定子在馬達上交換速率的時間尺度會些微上升。而追蹤蛋白在膜上擴散的速率顯示，FliL與定子在膜上的功能應該是各自獨立。我們的這些結果表明了FliL對於定子在馬達上的功能具有輔助定子的功能，並提出了可能的組裝過程；第三個計畫主要利用在大腸桿菌上的嵌合體馬達，探討環境中鈉離子濃度對於定子在馬達上組裝動力學的相關反應。我們發現鈉驅動的嵌合體馬達上，定子的數目會有所調整以因應鈉離子驅動勢的變化，因此可以在很寬的鈉離子濃度範圍內，保持一個穩定的速度。根據量測，馬達在鈉離子濃度5mM下，定子在馬達上的結合最為穩定，高於其他更高的鈉離子濃度條件。並且，當環境中鈉離子濃度快速的從高濃度切換到低濃度時，馬達上運作的定子數目出現先快速的下降再回升的現象，該結果與現行用來預測定子組裝過程的吸收模型有所不同，這樣的結果暗示著，當鈉離子濃度突然間的下降，定子可能進入了尚未了解的亞穩態。總結來說，這本論文中應用了複合式顯微術，結合電腦控制的流道系統，量測了鞭毛馬達的轉速與定子螢光實驗，提供了一個前沿的系統從不同角度綜合量測蛋白質的動力學。我們對鈉驅動鞭毛馬達的發現凸顯了生物分子複雜的動態機制，並且為更深入研究鞭毛馬達的運作機制提供了可能性。 ;Bacterial flagellar motor (BFM) is a nanometer-size molecular machine (~50 nm) in many bacteria species. By rotating the flagellar motor, bacteria have the motility to search life essentials actively at low Reynold number environment. A flagellar motor composes of a helical flagellum, a rotor and about dozen of stator units. A rotor is a rotary body connecting to a flagellum. Stator units are small independent protein complex that drive the rotor rotation and consume ion-motive force by transporting specific ions (mostly H+ or Na+) into cytoplasmic to drive the rotor rotation. Moreover, stator units dynamically associate/dissociate at the circumference of a functional rotor. The stoichiometry of stator units in a functional motor is highly dynamic and imperative to the power output. Therefore, understanding the assembly kinetics of stator units is crucial to elucidate bacteria flagellar motor′s the primary working mechanism and characteristics. The work in this thesis focuses mainly on sodium-type stator units, including wild-type stator in Vibrio alginolyticus and chimeric stator in Escherichia coli. We applied multiple techniques of optical microscopy to reveal the stoichiometry and kinetic of stator units, including total internal reflection fluorescence microscopy (TIRF), fluorescence recovery after photobleaching (FRAP), photoactivated localization microscopy (PALM), and back focal plane interferometry (BFPI). These techniques were designed and integrated into a commercial microscope. The apparatus was further devised to perform an on-time perfusion system for ion concentration switching and a focus-lock system for long-term focus stability. In this thesis, three related projects are presented. The first project aims to investigate the distribution of stator units around a motor by PALM. The estimated spatial resolution is smaller than 5 nm using PALM in our system. We applied two-color labelling on both rotor protein FliN and stator protein PomA for the stator localization experiments. After we carefully examed the colocalization ratio and the fixation effect, we found the stator density is a constant near the rotor and decayed with distance. However, the stator number density is lower than the model. Further investigation is required for the PALM experimental system. The second project aims to analyze the affection of torque association protein, FliL, to stator-units assemble in V. alginolyticus. Using TIRF microscopy to count protein quantity, the stator units and FliL protein stoichiometric ratio is close to 1:1 in a functional motor. Besides, the exchange time of stator units is slightly increased in the absence of FliL from the FRAP experiment. Measurement of the diffusion rate suggests the two proteins are independent on the membrane. Our results suggest that FliL plays a supporting role to the stator in the BFM. The third project investigates the relation between sodium concentration and the kinetic response of chimaeric stator units in E. coli. We found the sodium-driven chimaeric BFMs maintained constant speed over a wide range of sodium concentrations by adjusting stator units in compensation to the sodium-motive force (SMF) changes. The BFM has the maximum number of stator units and is most stable at 5 mM sodium concentration rather than higher sodium concentration. Upon rapid exchange from high to low sodium concentration, the stator number shows a drop and then resurrection that is different from predictions of simple absorption model. This may imply the existence of a metastable hidden state of the stator unit during the sudden loss of sodium ions. In conclusion, our correlated microscopy combining BFM speed and fluorescence measurements with computer-controlled microfluidic devices provided a cutting-edge system to investigate protein complex dynamics. Our findings of sodium-driven BFMs highlight the complex and dynamical mechanism of biomolecules and pave the way of the complete understanding of BFM working mechanisms.
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