熒光光譜在蛋白質(zhì)研究中的應(yīng)用課件

1、熒光光譜在蛋白質(zhì)研究中的應(yīng)用 一、基本原理 1、熒光的產(chǎn)生2、熒光的壽命：10-810-4 s3、磷光的壽命： 10-4102 s4、紫外的壽命： 10-15 s 紫外觀察不到的過程，其它兩種光譜可以觀察到，如生色團(tuán)及其環(huán)境的變化。5、由于它是發(fā)射光譜，所以靈敏度很高。6、磷光 二、儀器結(jié)構(gòu)與主要光譜參量1、儀器結(jié)構(gòu)2、主要光譜參量（1）吸收譜、熒光激發(fā)光譜、及熒光發(fā)射光譜 N-甲基咔唑在環(huán)己烷（2）熒光壽命 去掉激發(fā)光后，分子的熒光強(qiáng)度(I0)將到1/e I0所需要的時(shí)間，用表示， = 1/k。如果激發(fā)態(tài)分子只以發(fā)射熒光的方式丟失能量，熒光壽命稱為F, 它與kF（熒光發(fā)射速率常數(shù)）成反比，

2、F可以給出有關(guān)分子結(jié)構(gòu)和動(dòng)力學(xué)方面的信息。（3）量子產(chǎn)率 表示物質(zhì)發(fā)射熒光的效率，用表示。發(fā)射熒光分子在全部退激分子中所占比例。（5）溶劑的影響 極性增加，發(fā)生紅移。（1-氨基-8-萘磺酸酯），從辛醇乙二醇，紅移且強(qiáng)度降低。（6）熒光淬滅因素 a. 碰撞淬滅 b. 能量轉(zhuǎn)移 c. 氧的淬滅作用 d. 自淬滅 e. 光照反應(yīng)計(jì)量比的測(cè)定反應(yīng)動(dòng)力學(xué)3. Zn2+對(duì)氨基?；傅挠绊? 熒光降低且紅移, Trp從疏水內(nèi)部向外部移動(dòng).4. 外源熒光的應(yīng)用5、共振能量轉(zhuǎn)移熒光共振能量（FRET）轉(zhuǎn)移及應(yīng)用輻射能量轉(zhuǎn)移 熒光分子發(fā)射的熒光為某一物質(zhì)分子吸收，前者（能量給予體,D）的熒光減弱或猝滅，后者（能量

3、接受體, A）被激發(fā)： D* D + A + A* 非輻射能量轉(zhuǎn)移 根據(jù)作用機(jī)理的不同，非輻射能量轉(zhuǎn)移分為共振能量轉(zhuǎn)移和交換能量轉(zhuǎn)移. 共振能量轉(zhuǎn)移即熒光共振能量轉(zhuǎn)移，機(jī)理是通過分子的偶極偶極耦合作用進(jìn)行能量轉(zhuǎn)移，分子間有一定的距離。能量傳遞距離甚至可達(dá)7.010.0nm,也稱為長(zhǎng)距離能量轉(zhuǎn)移.給予體和接受體的共振能量轉(zhuǎn)移能級(jí)圖RO值的計(jì)算 A1/2表示當(dāng)有50的能量轉(zhuǎn)移效率（即激發(fā)態(tài)給予體的50由熒光共振能量轉(zhuǎn)移去活化）.熒光共振能量轉(zhuǎn)移的基本條件給予體和接受體分子必須靠近（一般是1-10 nm），即在遠(yuǎn)大于碰撞半徑（r）的距離上發(fā)生。接受體的吸收光譜必須與給予體的熒光發(fā)射光譜重疊。給予體和

4、接受體分子的偶極轉(zhuǎn)移空間定向必須近似平行。能量轉(zhuǎn)移效率與介質(zhì)的粘度變化無關(guān)。最可能的熒光共振能量轉(zhuǎn)移是單重態(tài)-單重態(tài)和三重態(tài)-單重態(tài)能量轉(zhuǎn)移過程四甲基羅丹明碘乙酰胺菁染料Cy55-(2-IODOACETYL)AMINO)ETHYL)AMINO)NAPHTHALENE-1-SULFONIC ACID, IAEDANS5-(碘乙酰)氨基乙基氨基萘1-磺酸Msx(Msx-homeodomain) The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP

5、)-4 signal and a key regulator for neural tissue differentiation. 接受體分子是,標(biāo)記蛋白質(zhì)中6，10，27位置的半胱氨酸；給予5-(碘乙酰)氨基乙基氨基萘1-磺酸（IAEDANS）體是蛋白質(zhì)分子中48位置的色氨酸（Trp-48）,能量轉(zhuǎn)移發(fā)生在AEDANS和Trp-48之間，據(jù)此計(jì)算在蛋白質(zhì)中6，10，27位置到48位置的距離分別約是1.9，2.3和1.6 nm.研究型和突變體型胞漿谷胱甘肽轉(zhuǎn)移酶的區(qū)別 通過用1-苯胺基8萘磺酸（ANS）為接受體標(biāo)記胞漿谷胱甘肽轉(zhuǎn)移酶的非催化位點(diǎn)，以胞漿谷胱甘肽轉(zhuǎn)移酶中的催化位點(diǎn)色氨酸（Trp）

6、殘基為給予體，利用FRET研究人類型和突變體型胞漿谷光甘肽轉(zhuǎn)移酶的區(qū)別。發(fā)現(xiàn)，型中給予體-接受體間距離2.2 nm, 型中距離1.82 nm。 Application of FRET to the GroELGroES Chaperonin Reaction, GroEL (EL315C + IAEDANS) EL513-D, GroES(ES98C + F5M) ES98-AMETHODS 24, 278288 (2001)6、綠色熒光蛋白 綠色熒光蛋白空間結(jié)構(gòu) (Green Fluorescence Protein): 1962年在水母中分離得到。它由11個(gè)-折疊圍繞成一個(gè)-折疊桶，結(jié)構(gòu)中

7、唯一的一個(gè)-螺旋在桶的中間。 The mature protein resists many proteases and is stable up to 65C and pH 5 to 11, in 1% sodium dodecyl sulfate or 6M guanidiniam chloride. Fluorescent is optimal at pH 7.2 to 8.0. It requires only UV or blue light and oxygen for fluorescence without any cofactors or substrates. -螺旋包括一

8、個(gè)氨基酸三聯(lián)體：residues 65-67, Thr-Tyr-Gly，被埋在疏水的蛋白質(zhì)中間可以防止氧以及溶劑對(duì)熒光的淬滅 。a圖為GFP; b是一種從珊瑚中發(fā)現(xiàn)的紅色熒光蛋白 DsRed 。 GFP has a number of amazing properties that enable its use for in vivo imaging. Firstly, GFP can be expressed in a variety of cells, where it becomes spontaneously fluorescent without the aid of a cofac

9、tor. Secondly, GFP can be fused to a host protein to create a fusion protein that usually retains both the fluorescence of the GFP and the biochemical function of the original host. Thirdly, fusion proteins can be targeted to specific organelles, such as the nucleus or endoplasmic reticulum, by addi

10、ng an appropriate signaling peptide. Finally, and most importantly, mutagenesis of GFP has produced many mutants with varying spectral properties that can be used as donors and acceptors of FRET.Activation of intracellular proteases such as caspase is an important event in programmed cell death. It

11、has been demonstrated that tumor necrosis factor (TNF), Fas ligand and chemotherapeutic drugs, are able to induce apoptosis by activating caspase.Caspase activation and proteolytic cleavage of specific target proteins represents an integral step in the pathway leading to theapoptotic death of cells.

12、 Analysis of caspase activity in intact cells, however, has been generally limited to the measurement of end-point biochemical and morphological markers of apoptosis.Substrates of group II caspase share a consensus recognition and cleavage amino acid sequence: DXXD. Detection of caspase activation i

13、n live cells will greatly facilitate monitoring of the apoptosis process in live cells. In an effort to develop a strategy with which to monitor caspase activity, early in the cell death cascade and in real-time, we have generated cell lines that overexpress recombinant GFP-based caspase substrates

14、that display a quantifiable change in their spectral properties when cleaved by group II caspases. In principle, caspase-3-mediated cleavage of these substrates should prevent fluorescence resonance energy transfer (FRET) between linked GFP proteins due to physical separation of the molecules. As the length of the li