荧光共振能量转移

1、荧光共振能量转移技术Fluorescence Resonance Energy Transfer（ FRET）MF11300191Why?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。2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31.1 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

2、供体,却能检测到受体发射的荧光41.1 FRET原理C=f51.2 FRET实现条件61.3 FRET探针/荧光集团荧光蛋白有机荧光染料镧系元素量子点 71.4 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82.文献学习DNA and nucleosomes direct distinct fold

3、ing of a linker histone H1 C-terminal domain He Fang1, David J. Clark2 and JeffreyJ.Hayes1*9 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 intrinsica

4、lly 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

5、 the condensed state.10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11Figure 1. The H1 CTD is intrinsically disordered and condenses upon binding to nucleosomes. ( A) Schematics o

6、f 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 (

7、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 C

8、y3/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?12Figure 2. Binding of C

9、y3/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

10、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

11、 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