熒光共振能量轉(zhuǎn)移

1、熒光共振能量轉(zhuǎn)移技術(shù)Fluorescence Resonance Energy Transfer（ FRET）MF11300191Why?研究蛋白質(zhì)與蛋白質(zhì)之間的相互作用Why？Why？酵母雙雜交、磷酸化抗體、免疫熒光、放射性標(biāo)記等方法應(yīng)用的前提都是要破碎細(xì)胞或?qū)?xì)胞造成損傷，無法做到在活細(xì)胞生理?xiàng)l件下實(shí)時(shí)的對(duì)細(xì)胞內(nèi)蛋白質(zhì)-蛋白質(zhì)間相互作用進(jìn)行動(dòng)態(tài)研究。2目錄1 FRET現(xiàn)象1.1 FRET原理1.2 FRET實(shí)現(xiàn)條件1.3 FRET探針1.4 FRET應(yīng)用2 文獻(xiàn)學(xué)習(xí)31.1 FRET現(xiàn)象1. 供體(Donor, D)2. 受體(Acceptor, A)3. FRET現(xiàn)象的特征: 選擇性激發(fā)

2、供體,卻能檢測到受體發(fā)射的熒光41.1 FRET原理C=f51.2 FRET實(shí)現(xiàn)條件61.3 FRET探針/熒光集團(tuán)熒光蛋白有機(jī)熒光染料鑭系元素量子點(diǎn) 71.4 FRET應(yīng)用活細(xì)胞生理?xiàng)l件下實(shí)時(shí)的對(duì)細(xì)胞內(nèi)蛋白質(zhì)-蛋白質(zhì)間相互作用進(jìn)行動(dòng)態(tài)研究定時(shí)、定量、定位、無損傷檢測活細(xì)胞內(nèi)蛋白質(zhì)-蛋白質(zhì)間相互作用1. 檢測酶活性變化（1）活細(xì)胞內(nèi)檢測蛋白激酶活性（2）關(guān)于細(xì)胞凋亡的研究2、關(guān)于膜蛋白的研究（1）受體激活效應(yīng)在細(xì)胞膜上的橫向擴(kuò)散（2）膜蛋白的定位修飾3、細(xì)胞膜受體之間相互作用4、細(xì)胞內(nèi)分子之間相互作用82.文獻(xiàn)學(xué)習(xí)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處理The protein was labeled with either maleimido-Cy3 or maleimido-Cy5, or a 50/50 mix of both according to manufacturers instructions (Pierce)Maleimido（馬來酰亞胺 ） ，一種熒光染料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