2023年 06期

嗜热光合酸杆菌Fenna-Matthews-Olson蛋白的分离、纯化与结晶

Isolation, Purification, and Crystallization of Fenna-Matthews-Olson Protein from Chloracidobacterium thermophilum


摘要(Abstract):

为了获得可用于X射线衍射分析的嗜热光合酸杆菌(Chloracidobacterium thermophilum,C.thermophilum)Fenna-Matthews-Olson (FMO)蛋白晶体,进一步探究FMO蛋白结构和功能,依次采用高浓度碳酸钠溶液浸泡、蔗糖密度梯度离心和二乙基氨基乙基葡聚糖凝胶阴离子交换层析方法,直接从嗜热光合酸杆菌中分离、纯化FMO蛋白;利用凝胶过滤层析及十二烷基磺酸钠-聚丙烯酰胺凝胶电泳,对该蛋白的均一性和纯度进行检测;分别使用坐滴法和悬滴法对蛋白结晶条件进行筛选和优化,并对优化的蛋白晶体进行X射线衍射分析。结果表明:制得的嗜热光合酸杆菌FMO蛋白纯度较高,性质均一;当温度为16℃、FMO蛋白质量浓度为15.0 g/L、池液中乙酸铵浓度为0.25 mol/L、缓冲液三羟甲基氨基甲烷-氯化氢浓度为0.1 mol/L、 pH为9.0,以及2-丙醇体积分数为32%时,可获得质量较好且最大衍射分辨率为0.235 nm的嗜热光合酸杆菌FMO蛋白晶体。

关键词(KeyWords): 生物学;蛋白质;嗜热光合酸杆菌;分离;纯化;结晶;

基金项目(Foundation): 国家自然科学基金项目(32200206,32270260); 泰山学者青年专家计划项目(tsqn201812079); 山东省自然科学基金项目(ZR2019ZD48,ZR2020QC057); 济南市高校院所科研带头人工作室项目(2020GXRC058);

作者(Author): 王佳佳,栾福琛,马林,董士尚秦晓春

DOI: 10.13349/j.cnki.jdxbn.20230403.002

参考文献(References):

[1]THIEL V, TANK M, BRYANT D A. Diversity of chlorophototrophic bacteria revealed in the omics era[J]. Annual Review of Plant Biology, 2018, 69(1): 21.

[2]THWEATT J L, CANNIFFE D P, BRYANT D A. Biosynthesis of chlorophylls and bacteriochlorophylls in green bacteria[J]. Advances in Botanical Research, 2019, 90: 35.

[3]BRYANT D A, FRIGAARD N-U. Prokaryotic photosynthesis and phototrophy illuminated[J]. Trends in Microbiology, 2006, 14(11): 488.

[4]TSUJI J M, SHAW N A, NAGASHIMA S, et al. Anoxygenic phototrophic chloroflexota member uses a type I reaction center[J]. Molecular Biology and Evolution, 2020, 23 (11): 2001-2007.

[5]GOLBECK J H. Shared thematic elements in photochemical reaction centers[J]. Proceedings of the National Academy of Sciences of the United States of America, 1993, 90 (5): 1642-1646.

[6]BRYANT D A, GARCIA COSTAS A M, MARESCA J A, et al. Candidatus Chloracidobacterium thermophilum: an aerobic phototrophic Acidobacterium[J]. Science, 2007, 317(5837): 523-526

[7]ZENG Y H, FENG F Y, HAHA M, et al. Functional type 2 photosynthetic reaction centers found in the rare bacterial phylum Gemmatimonadetes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(21): 7795-7800.

[8]ORF G S, GISRIEL C, REDDING K E. Evolution of photosynthetic reaction centers: insights from the structure of the heliobacterial reaction center[J]. Photosynthesis Research, 2018, 138(1): 11-37.

[9]ALLEN J P, WILLIAMS J C. Photosynthetic reaction centers[J]. FEBS Letters, 1998, 438(1/2): 5-9.

[10]ORF G S, BLANKENSHIP R E. Chlorosome antenna complexes from green photosynthetic bacteria[J]. Photosynth Research. 2013, 116(2/3): 315.

[11]FRIGAARD N-U, BRYANT D A. Chlorosomes: antenna organelles in photosynthetic green bacteria [M]//JESSUP M S. Complex Intracellular Structures in Prokaryotes. Berlin:Springer-Verlag, 2006: 89.

[12]WEN J Z, ZHANG H, GROSS M L, et al. Membrane orientation of the FMO antenna protein from Chlorobaculum tepidum as determined by mass spectrometry-based footprinting[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(15): 6134-6139.

[13]FENNA R E, MATTHEWS B W, OLSON J M, et al. Structure of a bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola: crystallographic evidence for a trimer[J]. Journal of Molecular Biology, 1974, 84(2): 231-240.

[14]FENNA R E, MATTHEWS B W. Chlorophyll arrangement in bacteriochlorophyll protein from Chlorobium limicola[J]. Nature, 1975, 258: 573-577.

[15]MATTHEWS B W, FENNA R E, BOLOGNESI M C, et al. Structure of a bacteriochlorophyll a-protein from the green photosynthetic bacterium Prosthecochloris aestuarii[J]. Journal of Molecular Biology, 1979, 131(2): 259-285.

[16]TRONRUD D E, WEN J, GAY L, et al. The structural basis for the difference in absorbance spectra for the FMO antenna protein from various green sulfur bacteria[J]. Photosynthesis Research, 2009, 100(2):79-87.

[17]CHEN J H, WU H J, XU C H, et al. Architecture of the photosynthetic complex from a green sulfur bacterium[J]. Science, 2020, 370 (6519): 1-8.

[18]WEN J Z, TSUKATANI Y, CUI W D, et al. Structural model and spectroscopic characteristics of the FMO antenna protein from the aerobic chlorophototroph, Candidatus Chloracidobacterium thermophilum[J]. Biochimica et Biophysica Acta, 2010, 1807(1): 157-164.

[19]TSUKATANI Y, WEN J Z, BLANKESHIP R E, et al. Characterization of the FMO protein from the aerobic chlorophototroph, Candidatus Chloracidobacterium thermophilum[J]. Photosynthesis Research, 2010, 104(2/3): 201-209.

[20]WHITMAN W B. Bergey's manual of systematics of archaea and bacteria[M]. New York: John Wiley Sons, 2015.

[21]TANK M, THIEL V, WARD D M, et al. A panoply of phototrophs: an overview of the thermophilic chlorophototrophs of the microbial mats of alkaline siliceous hot springs in Yellowstone National Park, WY, USA[M]//PATRICK C H. Modern Topics in the Phototrophic Prokaryotes. Switzerland: Springer, Cham, 2017: 87-137.