荧光共振能量转移

,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2011/12/21,#,荧光共振能量转移技术,F,luorescence,R,esonance,E,nergy,T,ransfer,（,FRET,）,王伟,MF1130019,Why?,研究蛋白质与蛋白质之间的相互作用,Why,？,Why,？,酵母双杂交、磷酸化抗体、免疫荧光、放射性标记等方法应用的前提都是要破碎细胞或对细胞造成损伤，无法做到在活细胞生理条件下实时的对细胞内蛋白质,-,蛋白质间相互作用进行动态研究。,目录,1 FRET,现象,1.1 FRET,原理,1.2 FRET,实现条件,1.3 FRET,探针,1.4 FRET,应用,2,文献学习,1.1 FRET,现象,1.,供体,(,Donor,D,),2.,受体,(Acceptor,A),3.FRET,现象的特征,:,选择性激发供体,却能检测到受体发射的荧光,1.1 FRET,原理,C=f,1.2 FRET,实现,条件,1.3 FRET,探针,/,荧光集团,荧光,蛋白,有机,荧光染料,镧系元素,量子点,1.4 FRET,应用,活细胞,生理条件,下实时的对,细胞内蛋白质,-,蛋白质间相互作用进行,动态,研究,定时、定量、定位、无损伤检测活细胞内,蛋白质,-,蛋白质,间相互作用,1.,检测,酶活性变化,（,1,）活细胞内检测蛋白激酶活性,（,2,）关于细胞凋亡的,研究,2,、关于膜蛋白的研究,（,1,）受体激活效应在细胞膜上的横向扩散,（,2,）膜蛋白的定位,修饰,3,、细胞膜受体之间相互作用,4,、细胞内分子之间相互作用,2,.,文献学习,DNA,and nucleosomes direct distinct,folding of,a linker histone H1 C-terminal,domain,He Fang,1,David J.,Clark,2,and JeffreyJ.Hayes1*,We,previously documented condensation of the,H1 CTD,consistent with adoption of a defined,structure upon nucleosome,binding using a bulk FRET,assay,supporting,proposals that the CTD behaves as,an intrinsically,disordered domain,.,In the,present study,by determining the,distances between two different,pairs of sites in the C-terminal domain,of full,length H1 by FRET,we confirm that,nucleosome binding,directs folding of the disordered,H1 C-terminal,domain and provide additional,distance constraints,for the condensed state.,处理,The protein was labeled with,either maleimido-Cy3,or maleimido-Cy5,or a 50/50 mix of,both according,to manufacturers instructions(Pierce),Maleimido,（,马,来酰,亚胺），一种荧光染料,Figure 1.The H1 CTD is intrinsically disordered and condenses upon binding to nucleosomes.,(A),Schematics of H1 G0101C/K195C(left)and,H1 G101C/T173C,(right)modified with Cy3 and Cy5(red and blue).N,G and C denote the N-terminal,globular and C-terminal domains,respectively,.,(,B),Binding of Cy3/Cy5-labeled H1 G101C/K195C to nucleosomes results in significant FRET.Emission spectra of 5 nM free H1(black line)and,H1 in,the presence of increasing amounts of 207 N nucleosomes,as indicated.Numbers indicate molar ratio of nucleosome:H1.Excitation was,at 515,nm.,(C),As in(B)except protein was Cy3/Cy5-labeled H1 G101C/T173C.,(D),Plot of FRET efficiency as function of nucleosome:H1 ratio,for Cy3/Cy5-labeled,H1 G101C/K195C and H1 G101C/T173C(filled black triangles and blue circles,respectively,Cy3/Cy5).Also shown are,efficiencies for,1:1 mixtures of Cy3-and Cy5-only labeled G101C/K195C and G101C/T173C(triangles and circles,respectively,Cy3+Cy5).,Inter?,Intra?,Figure 2.Binding of Cy3/Cy5-labeled H1 G101C/K195C to naked DNA results in both intra-and inter-molecular FRET.,(A),Binding of,Cy3/Cy5 labeled,H1 G101C/K195C to naked DNA induces FRET.The protein was incubated alone(black trace)or with increasing amounts of naked,207-bp DNA,fragments,as indicated.The molar ratio of DNA:H1 is indicated as is the concentration of DNA in microgram and microliter(in parenthesis,).,(,B),Intermolecular FRET upon H1 binding to naked DNA.A 1:1 mixture of Cy3-only and Cy5-only labeled H1 G101C/K195C was incubated,alone or,in the presence of increasing amounts of 207-bp DNA fragment as in A.,(C),Schematic of H1DNA tramtrack structure(24).H1 is indicated,by the,red ovals;DNA by the lines.,(D),Model for dilution of inter-molecular FRET with unlabeled H1(open ovals).,(E),Elimination of,inter-molecular FRET,to reveal intra-molecular FRET.Efficiencies for samples prepared as in A(H1-Cy3/Cy5)or B(H1-Cy3+H1-Cy5)and were determined,and plotted,(0 point)along with efficiencies for samples in which increasing fractions of the H1 was not labeled with fluorophores.,3Q!,