D97N突变对光受体蛋白古紫质4质子泵和能量转换效率的影响

doi:10.13523/j.cb.20170904

中国生物工程杂志

2017, Vol. 37

Issue (9): 23-30 DOI: 10.13523/j.cb.20170904

研究报告

D97N突变对光受体蛋白古紫质4质子泵和能量转换效率的影响

王娟, 郜玉娇, 孙超, 赵欣

华东师范大学物理与材料科学学院上海磁共振重点实验室上海 200062

Effect of D97N Mutation on Proton Transport and Energy Conversion in the Photoreceptor Archaerhodopsin 4

WANG Juan, GAO Yu-jiao, SUN Chao, ZHAO Xin

Shanghai Key Laboratory of Magnetic Resonance, School of Physics and Materials Science, East China Normal University, Shanghai 200062, China

全文: PDF(1155 KB) HTML

摘要： 古紫质4（archaerhodopsin 4，aR4）是新近发现的古生菌Halobacterium species xz515红膜上唯一的光敏视黄醛蛋白，具有和细菌视紫红质（Bacteriorhodopsin，bR）相似的质子泵功能，但在中性pH条件下，其质子释放和摄取顺序与bR相反。针对质子供体天冬氨酸97（Aspartic acid 97，D97）对aR4光循环；特别是对质子释放摄取顺序和菌株ATP生成率的影响，采用基因定点突变技术，构建了aR4的单突变体D97N，以及相对应的bR单突变体D96N。采用紫外-可见吸收光谱和闪光动力学光谱技术初步研究突变对视黄醛键合区、光反应中间态M态和O态、质子泵功能以及菌株ATP生成率的影响。结果表明，D97N突变对视黄醛紫外-可见光吸收波长没有太大影响，但造成M态衰减时间的显著延长、质子泵功能的消失及菌株ATP的生成率大幅降低。与bR中的D96作用相比，D97对aR4质子功能的影响有所不同，这可能与D97所处的一个更为疏水性的胞外侧环境有关。

关键词： 单点突变; 光循环中间态; 古紫质4; 质子泵; 天冬氨酸97

Abstract: Archaerhodopsin 4 (aR4) is a newly discovered photosensitive retinal protein from the claret membrane of Halobacterium species xz515. It functions as a proton pump similar to bacteriorhodopsin (bR) but with an opposite temporal order of proton uptake and release at neutral pH. Aspartic acid 97 (D97) is one of the key residues as the proton donor during the photocycle in aR4. Its function on the photocycle, the proton pump function and the energy conversion is very important for a deep understanding of the relationships between aR4 structure and function, especially with proton release and uptake order. Single mutation of D96N of bR and D97N of aR4 have been successfully constructed by combining site-directed mutagenesis with heterologous expression in Halobacterium species L33. The influences of mutations on the retinal binding pocket, transient kinetic change of the M state and O state, and the ATP conversion efficiency have been studied by UV-VIS and flashlight induced kinetic absorption change spectroscopy. The results show that D97N mutation in aR4 does not affect the UV absorption of the retinal chromophore, but results in a significant prolongation of the M state, a greatly weakened proton pump, and a decreased ATP formation efficiency. This indicates a different function of D97 in aR4 than the same type residue D96 in bR due to a more hydrophobic environment along the proton translocation channel from D97 to the retinal chromophore.

Key words: Aspartic acid 97 Site-specific mutation Proton pump Photo-intermediate Archaerhodopsin 4

收稿日期: 2017-02-21 出版日期: 2017-09-25

ZTFLH:

Q93

基金资助: 国家自然科学基金资助项目（21475045，30970657）

通讯作者: 赵欣 E-mail: xzhao@phy.ecnu.edu.cn

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引用本文:

王娟, 郜玉娇, 孙超, 赵欣. D97N突变对光受体蛋白古紫质4质子泵和能量转换效率的影响[J]. 中国生物工程杂志, 2017, 37(9): 23-30.

WANG Juan, GAO Yu-jiao, SUN Chao, ZHAO Xin. Effect of D97N Mutation on Proton Transport and Energy Conversion in the Photoreceptor Archaerhodopsin 4. China Biotechnology, 2017, 37(9): 23-30.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170904 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I9/23

[1] Oesterhelt D, Stoeckenius W. Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nature:New biology, 1971, 233(39):149-152.
[2] Mukohata Y, Sugiyama Y, Ihara K, et al. An Australian halobacterium contains a novel proton pump retinal protein:Archaerhodopsin. Biochemical and Biophysical Research Communications, 1988, 151(3):1339-1345.
[3] Sugiyama Y, Maeda M, Futai M, et al. Isolation of a gene that encodes a new retinal protein, archaerhodopsin, from Halobacterium sp. aus-1. Journal of Biological Chemistry, 1989, 264(35):20859-20862.
[4] Li Q G, Wang H, Song D J, et al. The isolation and purification of archaerhodopsin from Halobacterium sp. XZ515. Acta Biochimica et Biophysica Sinica, Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(5):517-520.
[5] Matsuno-Yagi A, Mukohata Y. Two possible roles of bacteriorhodopsin; a comparative study of strains of Halobacterium halobium differing in pigmentation. Biochemical and Biophysical Research Communications 1977, 78(1):237-243.
[6] Bogomolni R A, Spudich J L. Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium. PNAS, 1982, 79(20):6250-6254.
[7] Beja O, Aravind L, Koonin E V, et al. Bacterial rhodopsin:evidence for a new type of phototrophy in the sea. Science, 2000, 289(5486):1902-1906.
[8] Ernst O P, Lodowski D T, Elstner M, et al. Microbial and animal rhodopsins:structures, functions, and molecular mechanisms. Chemical Reviews, 2014, 114(1):126-163.
[9] Li Q, Sun Q, Zhao W, et al. Newly isolated archaerhodopsin from a strain of Chinese halobacteria and its proton pumping behavior. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2000, 1466(1-2):260-266.
[10] Ming M, Lu M, Balashov S P, et al. pH dependence of light-driven proton pumping by an archaerhodopsin from Tibet:comparison with bacteriorhodopsin. Biophysical Journal, 2006, 90(9):3322-3332.
[11] Cline S W, Lam W L, Charlebois R L, et al. Transformation methods for halophilic archaebacteria. Canadian Journal of Microbiology, 1989, 35(1):148-152.
[12] Luecke H, Schobert B, Richter H T, et al. Structure of bacteriorhodopsin at 1.55Å resolution. Journal of Molecular Biology, 1999, 291(4):899-911.
[13] Zimanyi L, Kulcsar A, Lanyi J K, et al. Intermediate spectra and photocycle kinetics of the Asp96-> Asn mutant bacteriorhodopsin determined by singular value decomposition with self-modeling. PNAS, 1999, 96(8):4414-4419.
[14] Zimanyi L, Cao Y, Chang M, et al. The two consecutive M substates in the photocycle of bacteriorhodopsin are affected specifically by the D85N and D96N residue replacements. Photochemistry and Photobiology, 1992, 56(6):1049-1055.
[15] Holz M, Drachev L A, Mogi T, et al. Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement. PNAS, 1989, 86(7):2167-2171.
[16] Walter J M, Greenfield D, Bustamante C, et al. Light-powering Escherichia coli with proteorhodopsin. PNAS, 2007, 104(7):2408-2412.
[17] Hampp N. Bacteriorhodopsin as a photochromic retinal protein for optical memories. Chemical Reviews, 2000, 100(5):1755-1776.
[18] Lanyi J K, Pohorille A. Proton pumps:mechanism of action and applications. Trends in Biotechnology, 2001, 19(4):140-144.

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