以嵌合蛋白質探討 HmBRI 第三胞內環之功能

Exploring the Function of the Third Cytoplasmic Loop in HmBRI Using Chimeric Proteins

林修平(Hsiu-Ping Lin)

指導教授 ： 楊啓伸

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01172

摘要

嗜鹽性古細菌的視紫紅質可依照其功能被分為四種不同的型態，分別為氫離子幫浦的細菌視紫紅質 (BR)、氯離子幫浦 (HR) 的氯視紫紅質及和正負光趨性相關的感光型視紫紅質 (SRΙ, SRII)。這些視紫紅質皆以 all-trans retinal 做為其發色基團，接受光照後產生異構化變成13-cis retinal 帶動視紫紅質產生結構上的變化而行使生理功能，而後再回到 all-trans retinal 等待下一次的活化。其中細菌視紫紅質、氯視紫紅質和負光趨性感光型視紫紅質的蛋白質結構已被解出，皆為具有七個穿膜α螺旋結構的膜蛋白質，然而這些基本結構相類似的蛋白質，卻具有截然不同的蛋白質特性和生理功能；其中細菌視紫紅質 (BR) 的功能為氫離子幫浦，將胞內的氫離子運送至胞外產生一氫離子之濃度梯度，驅動 ATP 合成酶合成 ATP 給細胞利用。利用結構軟體的分析及過去文獻的報導，我們推測位於第五個α螺旋 (E helix) 和第六個α螺旋 (F helix) 中間的彈性環狀區塊 (E-F loop) 很有可能關鍵性地影響細菌視紫紅質 (BR) 的功能和特性。本研究設計一系列的嵌合蛋白質，將嗜鹽性古細菌 Haloarcula marismortui 中細菌視紫紅質 HmBRΙ 的 E-F loop 置換成阿拉伯芥中 AtGCR1 的 E-F loop，並由大腸桿菌外源表現此一系列嵌合蛋白質，測試其特徵吸收峰及光驅動氫離子幫浦活性，發現這些不同長度的嵌合蛋白質皆保有和 HmBR1 相似的特徵吸收峰，光驅動氫離子幫浦的生理活性也依然存在，說明 HmBRΙ 的 E-F loop 在替換並延長了胺基酸序列後依然保留其原態的功能及維持正常的折疊。然而嵌合蛋白質額外存在有一相異於 HmBRI 之光吸收讀值，顯示 E-F loop 的置換對蛋白質造成某種程度的影響。藉由光週期的量測結果，發現嵌合蛋白質的光週期明顯地在中間態受到延遲，顯示 E-F loop 的置換影響了 HmBRI 自胞內獲取氫離子的過程，也同時證明了 E-F loop 在HmBRI 運送氫離子的機制中，至少扮演部份重要角色。
Chimera WT 是接上阿拉伯芥的 GPCR – AtGCR1 的 HmBRI，過去研究中，AtGCR1 的 E-F loop 是和其下游的 Gα – AtGPA1 進行交互作用的區域。本篇研究嘗試使用 GTP 類似物螢光劑 BODIPYTR-GTP 偵測 Chimera WT 與 AtGCR1 下游的 AtGPA1有無交互作用，以期建立一 GPCRs/Gα 交互作用的研究平台，彌補過去外源表現系統難以表現 GPCRs 的困境。然而，螢光劑易受到緩衝溶液中界面活性劑的干擾，受限於目前無法準確定量界面活性劑，螢光訊號的準確性仍有待評估。若能精確定量界面活性劑，以建構嵌合蛋白質為策略，使用螢光訊號進行 in vitro 的膜蛋白質與可溶蛋白質的交互作用測試，仍具有開發成為一藥物篩選平台的潛力。

關鍵字

嗜鹽性古細菌 ； 第三胞內環 ； 細菌視紫紅質

並列摘要

Light provides an important energy source for the biosphere on the Earth. Previous studies identified four types of photoreceptors belonging to the rhodopsin family based on different physiological functions in haloarchaea. Bacteriorhodopsin (BR) and halorhodopsin (HR) are known to be light-driven ion transporters while sensory rhodopsins (SRΙ, SRII) were concluded as a phototaxis mediator. These proteins are all the membrane proteins with seven trans-membrane α-helix structure. Even though sharing the similar basic structure, they play different physiological roles in cells. By comparing the features and characteristics of proteins structure, amino acid sequences and the literature reports, we speculate the third cytoplasmic loop (E-F loop) is likely critical in BR’s function. To study of the functional importance of E-F loop in bacteriorhodopsins, a series of chimeric HmBRI from Haloarcula marismortui with the EF loop replaced with various lengths of E-F loop residues from plant AtGCR1 were constructed. The chimeric proteins were over-expressed by Escherichia coli and the purified proteins showed no shift in the maximum absorbance and the proton pump activities were intact. However, the photocycle analysis shows that the chimeric protein delay in the intermediate state for about 2-times slower than HmBRI. Our results suggested the E-F loop significantly affect the characteristics of HmBRI by influencing the efficiency of proton uptake from cytoplasm. The functional importance of E-F loop in bacteriorhodopsin could possibly be established.
Previous study shows that the E-F loop of AtGCR1 interact with its downstream Gα - AtGPA1. In this study, the GTP analogue BODIPYTR-GTP was used as a fluorescent probe to detect the interaction between Chimera WT and AtGPA1. However, the detergent in buffer solution interferes with the fluorescent signal. Currently, there is no suitable method for accurate detergent quantification, so the fidelity of the fluorescence signal has yet to be confirmed. If the detergent can be accurately quantified, the strategy of using chimeric protein and fluorescent probe to monitor the interaction between GPCRs and Gα provides a potential approach for efficient in vitro drug screening.

並列關鍵字

Haloarcula marismortui ； third cytoplasmic loop ； bacteriorhodopsin

參考文獻

46 傅煦媛. 表現 Haloarcula marismortui 之六個光感受體揭露其獨特的感光特性 碩士 thesis, 國立台灣大學, (2008).

47 劉康正. Haloarcula marismortui 中 HmBRI 及 HmBRII 蛋白質特性及功能研究 碩士 thesis, 國立台灣大學, (2009).

1 Spudich, J. L., Yang, C. S., Jung, K. H. & Spudich, E. N. Retinylidene proteins: structures and functions from archaea to humans. Annu Rev Cell Dev Biol 16, 365-392, doi:10.1146/annurev.cellbio.16.1.36516/1/365 [pii] (2000).

2 Rao, V. R. & Oprian, D. D. Activating mutations of rhodopsin and other G protein-coupled receptors. Annu Rev Biophys Biomol Struct 25, 287-314, doi:10.1146/annurev.bb.25.060196.001443 (1996).

3 Lanyi, J. K. Progress toward an explicit mechanistic model for the light-driven pump, bacteriorhodopsin. FEBS Lett 464, 103-107, doi:S0014-5793(99)01685-3 [pii] (1999).