運輸鎂離子的視紫紅質之結構與光學特性研究

Abstract

古細菌、海洋細菌或是一些特定藻類足以存活於高光照、高鹽或是極端酸鹼值的環境中，而表現在這些生物細胞膜上的微生物視紫紅質 (microbial rhodopsin, mRho)，可以藉由吸收太陽光作為能量來源來執行他們多樣的功能。依照微生物視紫紅質的功能可以區分為兩大類：一類為光驅動離子幫浦，如向外運輸的氫離子幫浦 (bacteriorhodopsin, BR) 和向內運輸的氯離子幫浦 (halorhodopsin, HR)，另一類為感光型視紫紅質 (sensory rhodopsin, SR)，調控趨光或是避光的光趨性訊號。這些微生物視紫紅質被認為可以藉由本身的功能，來達成產生能量以供細菌使用、維持細胞滲透壓以抵抗外界溶劑以及調節光趨性訊號。近來許多證據顯示，微生物視紫紅質可以感知環境中因子的變化像是氯化鎂與氯化鈉濃度、酸鹼值、膜電位、滲透壓與壓力，從而影響其生理意義。本研究對一未知功能的微生物視紫紅質Middle rhodopsin (MR) 進行全方面探討，其被發現於鹽方扁平古菌 (Haloquadratum walsbyi, Hw)，此嗜鹽古細菌可以生存於高鹽且含有高濃度MgCl2的環境中 (2.0 M)。透過結構分析、光學特性分析與功能測試發現，MR可以作為一向內運輸鎂離子的轉運蛋白，在其結構中確定了兩個關鍵鎂離子結合位點，且其胺基酸D84、T216、D95個別扮演了離子選擇性、離子穩定、離子參與的關鍵位點。而位於蛋白質C端呈現靈活且高絲胺酸-蘇胺酸比例，極高可能與下游蛋白質結合進行訊號傳遞，光週期的結果可以支持此推測。由此我們提出了一個模型闡述了MR的鎂離子運輸機制。此外，因為在H. walsbyi基因組中BR與MR的近距離呈現出功能相關性，因此我們建構了一可以表現BR-MR融合蛋白的質體，並發現MR可以藉由幫助BR抵抗外界高濃度的鎂離子來維持質子運輸，最終達成生產能量供給細菌使用。在本研究中，我們以多面向的實驗方法來釐清MR向內運輸鎂離子的功能如何協助H. walsbyi生存。

Archaea, marine bacteria, and certain algae have demonstrated remarkable resilience in environments with high light intensity, salinity, or extreme pH levels. Within these organisms, microbial rhodopsins (mRho) are on cell membranes, harnessing sunlight to perform diverse functions. mRhos are classified into two primary categories based on their functions: light-driven ion pumps, such as outward proton pumps (bacteriorhodopsin, BR) and inward chloride pumps (halorhodopsin, HR), and sensory rhodopsins (SR), which mediate phototaxis signaling, facilitating either attraction or repulsion in response to light stimuli. The functions of mRhos are essential for energy generation, maintaining osmolarity against external solutes, and regulating phototaxis. Emerging evidences indicate that mRhos have the capability to sense changes in environmental factors like MgCl2 concentration, NaCl level, pH, membrane potential, osmolarity, and pressure, thereby influencing their physiological significances. Here, we conducted a comprehensive analysis of Middle rhodopsin (MR) derived from Haloquadratum walsbyi, a haloarchaeon surviving in a high-salinity environment with excessive MgCl2 concentrations (2.0 M). Through structural analysis, optical characterization, and functional assays, we determined that MR functioned as an inward Mg2+ transporter and identified two key Mg2+ binding sites. Specifically, residues D84, T216, and D95 were implicated in Mg2+ ion selectivity, stabilization, and association, respectively. Additionally, the flexible and high serine-threonine content in the C-terminus of MR implied potential protein interactions facilitating signal transduction, supported by photocycle kinetics. Our findings proposed a model elucidating the mechanism of Mg2+ transportation by MR. Furthermore, given the proximity of BR and MR in the genome of H. walsbyi, indicating their function correlation, we engineered a plasmid encoding a chimeric protein BR-MR. This construct demonstrated that MR aided BR in proton pumping against excessive Mg2+, leading to ATP production. In this study, we utilized a multifaceted approach to clarify the pivotal role of MR in the survival strategies of H. walsbyi, particularly through inward Mg2+ transportation mechanisms.