单分子荧光共振能量转移技术

单分子荧光共振能量转移技术（single molecule fluorescence resonance energy transfer,smFRET）通过检测单个分子内的荧光供体及受体间荧光能量转移的效率,来研究分子构象的变化。在单分子探测技术发展之前,大多数的分子实验是探测分子的综合平均效应（ensemble averages）,这一平均效应掩盖了许多特殊的信息。单分子探测可以对体系中的单个分子进行研究,得到某一分子特性的分布状况,也可研究生物分子的动力学反应。介绍了近来单分子荧光共振能量转移技术的进展。     

生物学中荧光共振能量转移的研究应用进展

荧光共振能量转移（FRET）可用于对生物大分子之间的距离进行定性、定量检测,所采用的材料、方法在近年都有了很大的发展,在核酸、蛋白质、细胞器结构功能检测、免疫测定、配体－受体相互作用测定等方面都有巧妙而有效的应用,应用前景十分广阔。     

用荧光共振能量转移研究synaptotagmin 胞质部分的寡聚化

目前人们公认synaptotagmin在神经递质释放过程中作为钙离子感受器而发挥作用。以前的研究发现,synaptotagmin存在两种形式的寡聚化,一种是通过跨膜区以及随后的中间链部分介导的寡聚化;另一种是通过胞质部分（C2AB）介导的寡聚化。对于后者有很多争议。在这篇文章中,作者用荧光共振能量转移的方法,在尽可能接近生理的条件下,证明了C2AB在有细胞膜和游离的钙离子的条件下能够寡聚化。而且,抽提细胞膜上的胆固醇或者封闭膜上的磷酸肌醇二磷酸能抑制C2AB在膜上的寡聚。     

利用荧光共振能量转移检测外源信号分子对拟南芥ATP水平的影响

本研究使用ATP特异性荧光共振能量转移(Fluorescence resonance energy transfer,FRET)为基础的荧光蛋白传感器(Ateam1.03-nD/nA),分析了4种外源信号分子(细胞外ATP、Ca²⁺、H₂O₂和NO)对拟南芥(Arabidopsis thaliana(L.)Heynh.)幼苗叶绿体和细胞质中ATP水平的影响。结果显示,细胞质ATP水平整体高于叶绿体,在4种不同浓度的信号分子处理下,叶绿体Ateam1.03-nD/nA的FRET比值仅在1.2 ~ 1.8波动;细胞质Ateam1.03-nD/nA 的FRET比值仅在2.2 ～ 3.0之间波动,未产生显著变化。结果表明在以上外源信号分子的作用下,植物细胞质和叶绿体ATP均维持在较为稳定的水平。     

荧光单分子检测技术在生命科学中的应用

荧光单分子检测技术是用荧光标记来显示和追踪单个分子的构象变化、动力学,单分子之间的相互作用以及单分子操纵的研究。过去对于生命科学分子机制的研究,都是对分子群体进行研究,然后平均化来进行单分子估测。因此,单个分子的动态性和独立性也被平均化掉而无法表现出来。荧光单分子检测技术真正实现了对单个分子的实时观测,将过去被平均化并隐藏在群体测量中不能获得的信息显示出来。近几年来,荧光单分子检测技术的飞速发展,为生命科学的发展,开辟了全新的研究领域。现就荧光单分子检测技术在研究动力蛋白、DNA转录、酶反应、蛋白质动态性和细胞信号转导方面的应用进展作一综述。     

生物大分子相互作用检测技术新进展———三色荧光级联荧光共振能量转移技术

荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET),是指能量从一种受激发的荧光基团(fluorophore)以非辐射的方式转移到另一种荧光基团的物理现象.FRET的能量转移效率是两个荧光基团间距离的函数,并对此距离十分敏感,它的有效响应距离一般在1～10nm之间,因而可被用于测定原子间及分子间的距离.这一特点使FRET技术在大分子构象变化、大分子之间相互作用、细胞信号通路等研究中发挥重要作用,成为生物医学研究中的重要方法.但细胞内的生物学过程常常涉及多于两个的大分子间相互作用,二色荧光基团的FRET技术不能满足这种生物学研究的需求.最近,两个研究小组在这方面取得突破,建立了分别基于共聚焦显微镜和流式细胞仪的三色荧光级联FRET技术.这一技术的出现将会极大地促进生物学及相关研究领域的发展.     

基于荧光蛋白的荧光共振能量转移探针的构建及应用

荧光共振能量转移(fluorescence resonance energy transfer,FRET)是基于荧光基团供体和荧光基团受体间偶极子–偶极子耦合作用的非辐射方式的能量传递现象。基于荧光蛋白的FRET技术已被广泛用于研究细胞信号通路中蛋白质–蛋白质活体相互作用检测、蛋白质构象变化监测以及生物探针的研制中。基于荧光蛋白的荧光共振能量转移探针使得人们可以在时间和空间层面上研究细胞信号的转导过程。该文简要介绍了四大类基于荧光蛋白的FRET生物探针的设计、研制以及其在生物信号分子检测、活细胞成像以及药物筛选中的应用和进展情况。     

小波变换及滚球算法在单分子荧光能量共振转移图像分析中的应用

单分子荧光共振能量转移技术是通过检测单个分子内的荧光供体及受体间荧光能量转移的效率来研究分子构象的变化．要得到这些生物大分子的信息就需要对大量的单分子信号进行统计分析,人工分析这些信息,既费时费力又不具备客观性和可重复性,因此本文将小波变换及滚球算法应用到单分子荧光能量共振转移图像中对单分子信号进行统计分析．在保证准确检测到单分子信号的前提下,文章对滚球算法和小波变换算法处理图像后的线性进行了分析,结果表明,滚球算法和小波变换算法不但能够很好地去除单分子FRET图像的背景噪声,同时还能很好地保持单分子荧光信号的线性．最后本文还利用滚球算法处理单分子FRET图像及统计15 bp DNA的FRET效率的直方图,通过计算得到了15 bp DNA的FRET效率值．     

基于量子点的荧光共振能量转移探针的应用

量子点因其独特的光学性质,以及可与有机分子所形成的偶联物的特殊性质,在光学生物标记,由其是荧光共振能量转移(Fluorescence resonance energy transfer,FRET)探针的合成与应用等领域具有广泛的应用前景,并因其实时、准确、灵敏的检测优势,在生物及医学领域始终被热切关注。该文以量子点的优势为基础,分别介绍了用于检测核酸、蛋白酶、生物反应及细胞状态的量子点-FRET探针的研究机理研究进展及应用优势。并对量子点-FRET探针的存在问题及研究方向进行了展望,为进一步进行该领域的研究提供理论支撑。     

利用荧光共振能量转移技术在活细胞内研究顺铂诱导的凋亡信号通路

为在活细胞内探讨顺铂诱导的凋亡通路．实验样品经顺铂处理后,应用基于荧光共振能量转移(FRET)原理设计的荧光探针pFRET-Bid和pSCAT-3来检测Bid切割和Capase-3活化的动态变化,同时,利用荧光成像在亚细胞水平对Bid转位线粒体的动力学特征进行了实时分析．结果表明：在顺铂诱导的细胞凋亡过程中,Bid切割发生在药物刺激后4～5 h, 历时(120±20) min．Bid切割活化后即从胞浆内转位到线粒体,历时(90±15) min．在凋亡后期,可以明显检测到Caspase-3 的激活．研究表明,应用FRET及荧光成像技术,可以在活细胞内实时、直观、可视地研究顺铂诱导的细胞凋亡过程,从而客观地反映了Bid、Caspase-3等蛋白质分子在该凋亡信号通路中的动态行为及时空传递特性．     

Ribosome degradation in growing bacteria

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.     

The Role of L1 Stalk-tRNA Interaction in the Ribosome Elongation Cycle

The ribosomal L1 stalk is a mobile structure implicated in directing tRNA movement during translocation through the ribosome. This article investigates three aspects of L1 stalk-tRNA interaction. First, by combining data from cryo electron microscopy, X-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different tRNAs occupying the hybrid P/E state interacting with the L1 stalk. These models confirm the assignment of fluorescence resonance energy transfer states from previous single-molecule investigations of L1 stalk dynamics. Second, the models reconcile how initiator tRNA^fMet interacts less strongly with the L1 stalk compared to elongator tRNA^Phe, as seen in previous single-molecule experiments. Third, results from a simulation of the entire ribosome in which the L1 stalk is moved from a half-closed conformation to its open conformation are found to support the hypothesis that L1 stalk opening is involved in tRNA release from the ribosome.     

smFRET studies of the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding

The selectivity with which a biomolecule can bind its cognate ligand when confronted by the vast array of structurally similar, competing ligands that are present in the cell underlies the fidelity of some of the most fundamental processes in biology. Because they collectively comprise one of only a few methods that can sensitively detect the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding, single-molecule fluorescence, and particularly single-molecule fluorescence resonance energy transfer (smFRET), approaches have revolutionized studies of ligand-binding reactions. Here, we describe a widely used smFRET strategy that enables investigations of a large variety of ligand-binding reactions, and discuss two such reactions, aminoacyl-tRNA selection during translation elongation and splice site selection during spliceosome assembly, that highlight both the successes and challenges of smFRET studies of ligand-binding reactions. We conclude by reviewing a number of emerging experimental and computational approaches that are expanding the capabilities of smFRET approaches for studies of ligand-binding reactions and that promise to reveal the mechanisms that control the selectivity of ligand binding with unprecedented resolution.     

罗伯逊易位研究的进展

本文总结了罗伯逊易位研究的现状、内容和技术方法,并对它在畜牧品种的改良、分子遗传学方面的应用作了探讨,指出了它在研究进化生物学方面的重要意义。 Abstract:Robertsonian translocation is important in the study of evolution biology.Current progress,contents,technology and methods about Robertsonian translocation studies are introduced in the article.The usefulness in improving the breeds of pasturages and molecular genetic aspect are also discussed.     

Mechanism of tRNA Translocation on the Ribosome

During the translocation step of the elongation cycle of peptide synthesis two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. Translocation is catalyzed by the elongation factor G (EF-G) and requires GTP hydrolysis. The fundamental biochemical features of the process were worked out in the 1970–80s, to a large part by A.S. Spirin and his colleagues. Recent results from pre-steady-state kinetic analysis and cryoelectron microscopy suggest that translocation is a multistep dynamic process that entails large-scale structural rearrangements of both ribosome and EF-G. Kinetic and thermodynamic data, together with the structural information on the conformational changes in the ribosome and EF-G, provide a detailed mechanistic model of translocation and suggest a mechanism of translocation catalysis by EF-G.     

A conformational change in the alpha-subunit of coatomer induced by ligand binding to gamma-COP revealed by single-pair FRET

Formation of transport vesicles involves polymerization of cytoplasmic coat proteins (COP). In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of γ-COP by the Golgi membrane protein p23 induces a conformational change that causes polymerization of the complex. Using single-pair fluorescence resonance energy transfer, we find that this conformational change takes place in individual coatomer complexes, independent of each other, and that the conformational rearrangement induced in γ-COP is transmitted within the complex to its α-subunit. We suggest that capture of membrane protein machinery triggers cage formation in the COPI system.