視黃醛結合袋中保守色胺酸點突變對兩種氫視紫質酸耐受性能力之不同影響

Mutations of Conserved Tryptophans in the Retinal Binding Pocket Contribute Differently upon Acid-tolerance in Two Different Bacteriorhodopsins

林宏軒(Hong-Syuan Lin)

指導教授 ： 楊啓伸

國立臺灣大學/生命科學院/生化科技學系/碩士(2018年)

https://doi.org/10.6342/NTU201802830

摘要

微生物視紫蛋白質 (microbial rhodopsin) 是一種廣泛分布在各種微生物細胞膜上的一種感光蛋白，此類蛋白質結構上含有七個穿膜的α螺旋，並在中心有一個視黃醛 (retinal) 與蛋白質第七穿膜區上離胺酸 (lysine) 鍵結形成希夫鹼 (Schiff base) ，構成發光基團 (chromophore)，而周邊接觸視黃醛並用以穩定發光基團結構的胺基酸稱為視黃醛結合口袋 (retinal binding pocket)，此微環境影響了不同蛋白質的吸收波長。在古生菌中，氫視紫質 (Bacteriorhodopsin, BR)是一種光驅動氫離子運輸蛋白，吸收特定波長光後，會將氫離子運輸至細胞膜外，提供ATP合成酶質動勢來產能。氫視紫質研究中最廣為了解的為Halobacterium salinarum的氫視紫質 (HsBR)，先前在HsBR的點突變研究中指出在視黃醛結合袋，會對發光基團的穩定性產生影響的胺基酸中，有相當高比例的色胺酸 (tryptophan)，也提出這些色胺酸在與視黃醛的交互作用中，扮演重要的角色。而在本實驗室中，有一種來自Haloquadratum walsbyi的氫視紫質 (HwBR)，相較與HsBR，在酸性環境下，發光基團穩定且可正常運輸氫離子。然而兩種BR在視黃醛結合袋的序列卻完全保守，卻對酸性環境有完全不同的穩定性。本研究想要去探討一樣的結構與序列卻有著如此之特性差異，是否包圍發光基團中保守的胺基酸，對於不同蛋白質而言其實扮演了不一樣的角色？本研究以先前研究指出有著重要性的色胺酸為對象，探討其在視紫質功能或是光學性質上所扮演的角色。本實驗中在HsBR和HwBR相同結構位置的的色胺酸做突變，並進行光學上與功能上的測量與分析，發現兩種BR序列保守的色胺酸對於蛋白質的光學和功能特性具有不一樣的影響，並提供色胺酸在可耐酸氫視紫質HwBR可能的功能，為往後發光基團對酸穩定性研究，提供一些基礎與線索。本實驗結果更顯示，對於序列分析為高度保守的胺基酸，仍需以更多和其他胺基酸交互作用來討論其作用。

關鍵字

氫視紫質 ； 視黃醛結合袋 ； 發光基團 ； 色胺酸 ； Haloquadratum walsbyi

並列摘要

Rhodopsins belong to a family of retinal-binding protein (RBP) which are widely distributed in different light sensitive organs. In microbes, rhodopsin are distributed on cell membrane, and they respond to light stimuli to mediate various physical functions. This protein family has a conserved seven-transmembrane domain with a retinal binding to a conserved lysine on the seventh helix. Retinal is further stabilized by highly conserved surrounding aromatic amino acids to form a retinal binding pocket, RBP. Bacteriorhodopsin (BR) is a light-driven outward proton pump found in haloarchaea, Upon light activation, BR pump a proton out of cell per photocycle to provide proton gradient to further ATP via F1Fo ATP synthase. Previous mutagenesis study in a well-studied bacteriorhodopsin, HsBR, showed that some residues in RBP can affect chromophore stability and the maximum absorbance (Abs-max). Among these residues, tryptophan residues were shown to play important roles in interaction with retinal and the Abs-max wavelength. Previously we found HwBR, a bacteriorhodopsin from Haloquadratum walsbyi, to have more stable Abs-max and functionality under acidic condition when compared to HsBR even though they have identical residues in RBP. To investigate such a paradox, this study mutated three corresponding tryptophan residues in both BR proteins and found conserved tryptophans contributed differently to the Abs-max and functionality under acidic conditions in two BR. We concluded further analysis in the interaction between tryptophan and nearby residues or water molecule network essential. This study also provide clear clues for future investigating on the acid-tolerance of RBP in microbial rhodopsin.

並列關鍵字

bacteriorhodopsin ； retinal binding pocket ； chromophore ； tryptophan ； Haloquadratum walsbyi

參考文獻

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