EF 環胺基酸單點突變改變 Halobacterium salinarum 古細菌視紫紅質之光電化學及動態吸光特性

Single Residue Mutations at the EF Loop Affect the Properties of the Photocurrent and Dynamic Absorbance Spectrum of Halobacterium salinarum Bacteriorhodopsin

鄭明晉(Ming-Jin Jheng)

指導教授 ： 楊啟伸

國立臺灣大學/生命科學院/生化科技學系/碩士(2013年)

https://doi.org/10.6342/NTU.2013.00575

摘要

古細菌視紫紅質 (Bacteriorhodopsin；BR) 為存在嗜鹽古細菌 Halobacterium salinarum 膜上的蛋白質，其為一光驅動氫離子幫浦 (light-driven proton pump)，照光後可將氫離子從細胞質打至細胞外。基態 BR 吸光後會依序轉變為各種中間過度態 (K、L、M、N 及 O)，行使其功能後，最後回到基態，這個照光後引發的循環稱為光週期。在 BR 的光週期中，氫離子會在 BR 內部的殘基間傳遞，也會在 BR 內部的殘基和外界的水之間傳遞。在 L 中間態轉變為 M 中間態的過程中，BR 內部的氫離子釋放複合體 (proton releasing complex；PRC) 會先釋放一個氫離子至細胞外，之後，在 N 中間態轉變為 O 中間態的過程中，Asp 96 再從細胞內獲得一個氫離子。因此，一個能快速偵測水溶液 pH 值變化的裝置，對研究 BR 釋放及獲得氫離子的速率是必要的。本研究的第一部分先利用兩個已知為氫離子獲得速率較慢的突變蛋白 (E161C 及 R164C)，確定了 BR-based 光電化學裝置在照光時的光電流衰減區 (decay of the light-on phase) 的衰減速率，以及停止照光後光電流恢復區 (recovery of the light-off phase) 的恢復速率，可以用來當作研究 wild type BR 及其突變蛋白獲得氫離子速率快慢的量化工具。而過去研究發現，BR 在行使功能的過程中 EF loop 會有顯著的構形變化，這表示 EF loop 很可能在 BR 行使功能時扮演了重要的角色。為了進一步研究 EF loop，本研究的第二部分將 EF loop 上的殘基單點突變為 Cys，並利用 BR-based 光電化學裝置和動態吸收光譜確定了 EF loop 上靠近 E-helix 及 F-helix 的 Cys 突變會顯著降低 BR 獲得氫離子的速率。這表示 EF loop 在 BR 獲得氫離子過程中扮演著重要的角色。

關鍵字

古細菌視紫紅質 獲得氫離子 BR-based 光電化學裝置 EF loop

並列摘要

Bacteriorhodopsin (BR) is the only protein in the purple membrane (PM) of Halobacteriun salinarum. BR is composed of seven transmembrane α-helices and contains one all-trans retinal chromophore which is covalently bound to the ϵ-amino group of the Lys 216 residue via a protonated Schiff base (PSB). Illumination of BR generates an electrochemical proton gradient across the PM by vectorial translocation of a proton from the cytoplasmic (CP) side to the extracellular (EC) side. Upon light absorption, the all-trans retinal isomerizes around the C13=C14 double-bond and reverts thermally back to the initial all-trans state, passing a series of intermediates named K, L, M, N, and O. The photocycle involves a proton transport process. During the L to M intermediate transition, a proton is released to the extracellular space from the proton releasing complex (PRC) which is composed of Arg 82, Glu 194, Glu 204, and internal H2O. During the N to O intermediate transition, the deprotonated Asp 96 uptake a proton from the cytoplasmic space. The transient pH change due to the light-induced proton release and uptake is usually studied by using a pH-sensitive dye whose absorption depends on pH. However, the restriction of the measurement is that the medium pH should be close to the pKa of the dye. Hence, a device is needed for the detection of the light-induced transient pH change in a wide pH range.
When illumination with a continuous wave (CW) laser pulse, a photocurrent is detected from the BR-based photoelectrochemical cell. At neutral pH, the positive photocurrent is observed when the CW light source is turned on, and the negative photocurrent is observed when the CW light source is turned off. The photocurrent is investigated for the transient proton concentration changes generated by BR. In previous study, it is found that the rates of the proton uptake decrease in the E161C and R164C mutant proteins using the pH-sensitive dye pyranine. In the first part of my work, I compare the photoresponse of the E161C and R164C mutations with the wild type BR. It is found that the decay rates of the light-on phase as well as the recovery rates of the light-off phase increase in the E161C and R164C mutations. These results suggest that the origin of the decay of the light-on phase as well as the recovery of the light-off phase are associated with the proton uptake of BR, and thus can be used for the investigation of the proton uptake rates of the wild type and mutant proteins. According to structural dynamic studies, significant conformational changes of the EF loop are observed during the photocycle of BR which means EF loop is likely critical in the function of BR. In the second part, I construct mutantions containing cysteine substitutions located in the EF loop of BR. Each of 10 residues, located in the EF loop of BR, was individually replaced by cystein, respectively. It is found that the rates of the proton uptake increase in the mutantions near the E-helix and F-helix. It means that EF loop play an important role during the proton uptake of BR.

並列關鍵字

Bacteriorhodopsin proton uptake BR-based photoelectrochemical cell EF loop

參考文獻

61 傅喣媛. 表現 Haloarcula marismortui 之六個光感受體揭露其獨特的感光特性. 國立台灣大學碩士論文 (2008).

63 沈宜中. HmBRI 突變設計蛋白質之光學特性與應用研究. 國立台灣大學碩士論文 (2012).

1 Rao, V. R. & Oprian, D. D. Activating mutations of rhodopsin and other G protein-coupled receptors. Annual review of biophysics and biomolecular structure 25, 287-314 (1996).

2 Spudich, J. L., Yang, C.-S., Jung, K.-H. & Spudich, E. N. Retinylidene proteins: structures and functions from archaea to humans. Annual review of cell and developmental biology 16, 365-392 (2000).

4 Oesterhelt, D. & Stoeckenius, W. Functions of a new photoreceptor membrane. Proceedings of the National Academy of Sciences 70, 2853-2857 (1973).