荧光光谱在蛋白质研究中应用.pptVIP

Application of FRET to the GroEL–GroES Chaperonin Reaction, GroEL (EL315C + IAEDANS) EL513-D, GroES(ES98C + F5M) ES98-A METHODS 24, 278–288 (2001) 6、绿色荧光蛋白 绿色荧光蛋白空间结构 (Green Fluorescence Protein): 1962年在水母中分离得到。它由11个?-折叠围绕成一个?-折叠桶，结构中唯一的一个?-螺旋在桶的中间。 The mature protein resists many proteases and is stable up to 65?C 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. ?-螺旋包括一个氨基酸三联体：residues 65-67, Thr-Tyr-Gly，被埋在疏水的蛋白质中间可以防止氧以及溶剂对荧光的淬灭 。a图为GFP; b是一种从珊瑚中发现的红色荧光蛋白 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 cofactor. 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 adding 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 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 the apoptotic death of cells. Analysis of caspase activity in intact cells, however, has been generally limited to the measurement of end-point biochemical and morphological markers of