比較兩關鍵結構區域對窩氏鹽方扁平古菌之HwBR其高抗酸性質之重要性

Abstract

對嗜鹽古生菌而言，氫視紫質會受光激發並作為氫離子幫浦，製造胞內外質子濃度梯度，進而驅動ATP合成酶運轉，對生產ATP相當重要。因此，如何在幫浦打出質子後，維持其功能不受外界提升後的質子濃度回饋抑制，為其能否達到更高的能源效率的關鍵。在2015年本實驗室分類的一種新BR亞型qR，發現到此類BR可以承受較低的pH環境，而窩氏鹽方扁平古菌 (Haloquadratum walsbyi) 的BR HwBR為其一員，同研究中亦已解得結構。鑑於BR是製造胞內外質子梯度來間接協助合成ATP，因此，可以耐胞外酸也表示，製造胞內外質子濃度差的能力是增加的。在先前實驗結果，發現到HwBR有較高的酸耐受性 (acid-tolerance)，且透過與結構比較，初步發現到有兩個區域與此性質有關，分別是在胞外側的R82與T201形成的脫水元 (dehydron) 結構，及在視黃醛結合口袋 (retinal binding pocket) D2位置的W94。在本研究中藉由將此三位點突變為R82E、T201S、W94F組合為三、雙、單點突變，藉由測試其光化學性質並比較不同突變組合的差異，進而了解各點位對HwBR酸耐受性之重要性。在光譜的實驗結果中，發現到W94F之有無決定了最高吸收峰 (Ab-max) 的藍移與否。在光電流的測試中，組合三突變點位後，蛋白質所能耐受酸的能力會因而下降，在pH 5.8的環境下便無法對抗環境中質子濃度進而打出質子，了解到三位點之間有抗酸的協同作用產生；而降低pH後，發現W94F突變株的酸耐受性明顯降低。進一步測試光週期狀態，在基態 (G state) 的結果中，W94F突變會造成光週期後段拖尾的現象；M態 (M state) 測試中，得到未受改變的結果；O態 (O state) 亦有明顯的拖尾，顯示前述W94F對BR作用機制造成的影響主要發生於O態。最後，本研究總結W94能獨立調節HwBR酸耐受性，然而BR整體仍仰賴胞外側的R82-T201及視黃醛結合口袋的W94共同合作，才能達到其高酸耐受性。

Bacteriorhodopsin is an important membrane protein of halobacteria for its light-driven property, working as a proton pump and causing a proton gradient to make ATP by ATP synthase. Therefore, the ability for BR to make proton gradient is directly related to the property of BR maintaining its function in increased extracellular proton concentrate. The higher proton concentration it stands, the more efficiently ATPs are produced. However, only qR, a newly cluster of BR subtype classified in 2015, can tolerate higher proton concentration including HwBR, BR from Haloquadratum walsbyi. In the same study, the structure of HwBR have been resolved. In previous studies, it showed that HwBR have higher acid tolerance and two regions in HwBR structure might be related to this property. Respectively, two regions are R82 and T201 in extracellular side which forming dehydron, and W94 at retinal binding pocket D2 site. This study investigates how rhodopsin structure affects the acid-tolerance especially in HwBR by mutating amino acids in two domains as R82E, T201S and W94F, to combine different mutant sites and test photochemical properties. In spectum and photocycle test, we found W94F is the main character makes red-shift and cycle delay. On the other side, we see signal revesed at pH 5.8 in photocurrent test with triple mutant, and acid tolerance of mutant W94F decrease when downgrading pH. Further, conducting photocurrent test, we find that W94F makes G state and O state extending but M state. This experiment explains that W94F influence O state mainly. In this study, we conclude that the two regions in BR actually have their own functions of avoiding extracellular protons affect BR function as a proton pump, and still the acid-tolerance property needs cooperation of .different structural regions.