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)
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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-~(15)N 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.
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-~(15)N 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.
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-~(15)N 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.
引文
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[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,K?STER 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,K?STER 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]K?STER T,MEYER K,WEINHOLDT C,et al.Regulation of pri-miRNA processing by the hnRNP-like protein At GRP7 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 m RNAs 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,SCH?NING 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].JBiomol 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 US 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,DANI?LS 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 WWdomain[J].Biochem Biophys Res Commun,2017,487(2):470-476.
[28]HOLM L,ROSENSTR?M P.Dali server:conservation mapping in 3D[J].Nucleic Acids Res,2010,38:W545-W549.
[29]SCH?NING 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.