拟南芥AtGrp7 RRM结构域的纯化及其结构与结合的初步分析(英文)

doi:10.11938/cjmr20182637

波谱学杂志 ›› 2019, Vol. 36 ›› Issue (1): 1-14.doi: 10.11938/cjmr20182637

拟南芥AtGrp7 RRM结构域的纯化及其结构与结合的初步分析(英文)

迟秀娟^1,2, 乔晓亚², 刘颖³, 刘惠丽⁴, 陈雷⁴, 王际辉¹, 艾选军²

1. 大连工业大学生物工程学院, 辽宁大连 116034;
2. 洁净能源国家实验室(中国科学院大连化学物理研究所), 辽宁大连 116023;
3. 国家艾滋病性病防治中心, 病毒与免疫研究室, 北京 102206;
4. 波谱与原子分子物理国家重点实验室, 武汉磁共振中心(中国科学院武汉物理与数学研究所), 湖北武汉 430071

收稿日期:2018-04-19 出版日期:2019-03-05 发布日期:2018-04-24
通讯作者: 王际辉,Tel:0411-86324050,E-mail:wangjh@dlpu.edu.cn;艾选军,Tel:0411-82463016,E-mail:xai@dicp.ac.cn E-mail:wangjh@dlpu.edu.cn;xai@dicp.ac.cn
基金资助:
The Liaoning Natural Science Foundation (20170520198, 20170520043); Project Supported by the State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics (T151601).

Purification of the AtGrp7 RRM Domain from Arabidopsis thaliana and Its Preliminary Structure and Binding Analysis

CHI Xiu-juan^1,2, QIAO Xiao-ya², LIU Ying³, LIU Hui-li⁴, CHEN Lei⁴, WANG Ji-hui¹, AI Xuan-jun²

1. School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China;
2. National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China;
3. Division of Virology & Immunology, National Center for AIDS/STD Control and Prevention, Beijing 102206, China;
4. State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan(Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences), Wuhan 430071, China

Received:2018-04-19 Online:2019-03-05 Published:2018-04-24
Supported by:
The Liaoning Natural Science Foundation (20170520198, 20170520043); Project Supported by the State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics (T151601).

摘要/Abstract

摘要： 富含甘氨酸的RNA结合蛋白AtGrp7是拟南芥（Arabidopsis thaliana）调节生物钟负反馈回路的组分.在使用常规方法纯化AtGrp7 RRM结构域的初始试验中，观察到烟草蚀纹病毒（TEV）酶切后AtGrp7 RNA识别基序（RRM）结构域的紫外吸收峰为蛋白和杂质的混合信号峰.为解决常规纯化中的杂质问题，对AtGrp7_1-90应用了变性-复性两步纯化方法.AtGrp7 RRM结构域的¹H-¹⁵N HSQC指纹谱和CS-Rosetta模型结构表明快速稀释重折叠后其结构完全恢复.等温滴定量热法（ITC）和核磁共振（NMR）滴定实验进一步证实，重折叠后AtGrp7_1-90 RRM结构域具有正确结合RNA/DNA的功能.

关键词: 变性-复性, 快速稀释, 核磁共振(NMR), AtGrp7 RNA识别基序(RRM)结构域, 结合分析

Abstract: The glycine-rich RNA-binding protein, AtGrp7, is a component of a negative feedback loop in the circadian clock regulation of Arabidopsis thaliana. In our initial purification trial of the tobacco etch virus (TEV)-cleaved AtGrp7 RNA recognition motif (RRM) domain with the regular protocol, mixed ultraviolet signals of the target proteins and contaminants were observed. A two-step denaturing-refolding protocol was then tested, trying to solve the problem of impurities. The structure of the AtGrp7_1-90 RRM domain was fully recovered by quick-dilution refolding, evidenced by the fingerprint ¹H-¹⁵N HSQC spectrum and CS-Rosetta model structures. Isothermal titration calorimetry (ITC) and NMR titration experiments further confirmed that the RRM domain of AtGrp7_1-90 had proper functions with regards to RNA/DNA binding.

Key words: denaturing-refolding, quick-dilution, nuclear magnetic resonance (NMR), AtGrp7 RNA recognition motif (RRM)domain, binding analysis

中图分类号:

O482.53

迟秀娟, 乔晓亚, 刘颖, 刘惠丽, 陈雷, 王际辉, 艾选军. 拟南芥AtGrp7 RRM结构域的纯化及其结构与结合的初步分析(英文)[J]. 波谱学杂志, 2019, 36(1): 1-14.

CHI Xiu-juan, QIAO Xiao-ya, LIU Ying, LIU Hui-li, CHEN Lei, WANG Ji-hui, AI Xuan-jun. Purification of the AtGrp7 RRM Domain from Arabidopsis thaliana and Its Preliminary Structure and Binding Analysis[J]. Chinese Journal of Magnetic Resonance, 2019, 36(1): 1-14.

参考文献

[1] HEINTZEN C, NATER M, APEL K, et al. AtGRP7, a nuclear RNA-binding protein as a component of a circadian-regulated negative feedback loop in Arabidopsis thaliana[J]. Proc Natl Acad Sci U S A, 1997, 94(16):8515-8520.
[2] STREITNER C, HENNIN L, KORNELI C, et al. Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7[J]. BMC Plant Biol, 2010, 10:221.
[3] MEYER K, KÖSTER T, NOLTE C, et al. Adaptation of iCLIP to plants determines the binding landscape of the clock regulated RNA-binding protein AtGRP7[J]. Genome Biol, 2017, 18(1):204.
[4] STREITNER C, KÖSTER T, SIMPSON C G, et al. An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana[J]. Nucleic Acids Res, 2012, 40(22):11240-11255.
[5] KÖSTER T, MEYER K, WEINHOLDT C, et al. Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis[J]. Nucleic Acids Res, 2014, 42(15):9925-9936.
[6] STREITNER C, DANISMAN S, WEHRLE F, et al. The small glycine-rich RNA binding protein AtGRP7 promotes floral transition in Arabidopsis thaliana[J]. Plant J, 2008, 56(2):239-250.
[7] KIM J S, JUNG H J, LEE H J, et al. Glycine-rich RNA-binding protein7 affects abiotic stress responses by regulating stomata opening and closing in Arabidopsis thaliana[J]. Plant J, 2008, 55(3):455-466.
[8] FU Z Q, GUO M, JEONG B R, et al. A type Ⅲ effector ADP-ribosylates RNA-binding proteins and quells plant immunity[J]. Nature, 2007, 447(7142):284-288.
[9] JEONG B R, LIN Y, JOE A, et al. Structure function analysis of an ADP-ribosyltransferase type Ⅲ effector and its RNA-binding target in plant immunity[J]. J Biol Chem, 2011, 286(50):43272-43281.
[10] NICAISE V, JOE A, JEONG B R, et al. Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs with GRP7[J]. EMBO J, 2013, 32(5):701-712.
[11] HACKMANN C, KORNELI C, KUTYNIOK M, et al. Salicylic acid-dependent and -independent impact of an RNA-binding protein on plant immunity[J]. Plant Cell Environ, 2014, 37(3):696-706.
[12] KIM J S, PARK S J, KWAK K J, et al. Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli[J]. Nucleic Acids Res, 2007, 35(2):506-516.
[13] WANG S C, LIANG D, SHI S G, et al. Isolation and characterization of a novel drought responsive gene encoding a glycine-rich RNA-binding protein in Malus prunifolia (Willd.) Borkh[J]. Plant Mol Biol Rep, 2011, 29(1):125-134.
[14] CAO S Q, JIANG L, SONG S Y, et al. AtGrp7 is involved in the regulation of abscisic acid and stress responses in Arabidopsis[J]. Cell Mol Biol Lett, 2006, 11(4):526-535.
[15] LEDER V, LUMMER M, TEGELER K, et al. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy[J]. Biochem Biophys Res Commun, 2014, 453(1):69-74.
[16] TRIPET B P, MASON K E, EILER B J, et al. Structural and biochemical analysis of the Hordeum vulgare L. HvGR-RBP1 protein, a glycine-rich rna-binding protein involved in the regulation of barley plant development and stress response[J]. Biochemistry, 2014, 53(50):7945-7960.
[17] FRANCO-ECHEVARRIA, GONZÁLEZ-POLO N, ZORRILLA S, et al. The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition[J]. Nucleic Acids Res, 2017, 45(17):10293-10305.
[18] SCHüTTPELZ M, SCHöNING J C, DOOSE S, et al. Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by Fluorescence correlation spectroscopy[J]. J Am Chem Soc, 2008, 130:9507-9513.
[19] DELAGLIO F, GRZESIEK S, VUISTER G W, et al. NMRPipe:a multidimensional spectral processing system based on UNIX pipes[J]. J Biomol NMR, 1995, 6(3):277-293.
[20] JOHNSON B A, BLEVINS R A. NMRView:a computer program for the visualization and analysis of NMR data[J]. J Biomol NMR, 1994, 4(5):603-614.
[21] LANGE O F, ROSSI P, SGOURAKIS N G, et al. Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples[J]. Proc Natl Acad Sci U S A, 2012, 109(27):10873-10878.
[22] SHEN Y, VEMON R, BAKER D, et al. De novo protein structure generation from incomplete chemical shift assignments[J]. J Biomol NMR, 2009, 43(2):63-78.
[23] SHEN Y, LANGE O, DELAGLIO F, et al. Consistent blind protein structure generation from NMR chemical shift data[J]. Proc Natl Acad Sci U S A, 2008, 105(12):4685-4690.
[24] FINN R D, COGGILL P, EBERHARDT R Y, et al. The Pfam protein families database:towards a more sustainable future[J]. Nucleic Acids Res, 2016, 44(D1):D279-D285.
[25] KHAN F, DANIËLS M A, FOLKERS G E, et al. Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein:implications for its RNA chaperone function[J]. Nucleic Acids Res, 2014, 42(13):8705-8718.
[26] HUTH J R, BEWLEY C A, JACKSON B M, et al. Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR[J]. Protein Sci, 1997, 6(11):2359-2364.
[27] QIAO X Y, LIU Y, LUO L T, et al. Effects of naturally occurring charged mutations on the structure, stability, and binding of the Pin1 WW domain[J]. Biochem Biophys Res Commun, 2017, 487(2):470-476.
[28] HOLM L, ROSENSTRÖM P. Dali server:conservation mapping in 3D[J]. Nucleic Acids Res, 2010, 38:W545-W549.
[29] SCHÖNING J C, STREITNER C, PAGE D R, et al. Auto-regulation of the circadian slave oscillator component AtGRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation[J]. Plant J, 2007, 52(6):1119-1130.

拟南芥AtGrp7 RRM结构域的纯化及其结构与结合的初步分析(英文)

Purification of the AtGrp7 RRM Domain from Arabidopsis thaliana and Its Preliminary Structure and Binding Analysis

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