第三章单分子生物物理学.ppt

1、Single Molecule Biophysics,23294,Single molecule,As of 2003 Single Molecules ceased publication and the articles are incorporated in ChemPhysChem,less,faster,Impetus to single molecule,What is single molecule biophysics?,Why we conduct single molecule or what single molecule can tell us?,How to stud

2、y the single molecules?,single molecules in diverse environments such as liquids, solids, membranes, surfaces, and polymers,Single Molecules provides a broad overview of current methods and techniques, recent applications and shortcomings of present techniques in the field. It covers all aspects of

3、single molecule investigations in chemistry, physics, and life sciences:,methods of manipulating and modifying single particles,single atoms and nanoparticles,all kinds of ultrasensitive detection techniques such as spectroscopies and microscopies,What is single molecule biophysics?,在单分子水平上对生物分子行为（包

4、括构象变化、相互作用、相互识别等）的实时动态检测以及在此基础上的操纵调控等，是分子生物物理学的自然延伸和必然趋势。生物单分子研究已成为21世纪生命科学领域的一个重点研究方向，在生命科学领域有着广泛的应用前景,Why we conduct single molecule or what single molecule can tell us?,In contrast with ensemble methods, single molecule experiments provide information on distributions and time trajectories of obs

5、ervables that would otherwise be hidden,single molecule methods,Sensitive or ultrasensitive,sensitivity,The photons detected from single molecules must exceed the background noise that originates from Raman and Rayleigh scattering and from fluorescence as a result of impurities in the solvent, glass

6、 coverslips, and optical components, and from dark current in the detector.,Genesis of noise?,a small excitation volume, to reduce the background,high-efficiency collection optics,detectors with high quantum efficiency and low dark noise,careful elimination of background fluorescence: solvent, optic

7、s, detection method et al.,How to increase S/N?,Techniques to study the single molecule,Laser Scanning Confocal Microscopy (LSCM),Total Internal Reflection Fluorescence Microscopy (TIRFM),Near field Scanning Optical Microscopy (NSOM or SNOM),Scanning Probe Microscopy(SPM):,Scanning Tunneling Microsc

8、ope STM,Atom Force Microscope ( AFM ),Optical tweezer/ optical trapOP or OT,A Single-Molecule Study of RNA Catalysis and Folding,The Nobel Prize in Physics 1997for development of methods to cool and trap atoms with laser light,Steven ChuUSA,Claude Cohen-TannoudjiFrance,William D. Phillips USA,在获得诺贝尔

9、奖的第二天，他骑着自行车，朝着目标往山路上攀爬，达到了目的地。这种攀登高峰的踏实感受:也只有在努力过之后，才能真切地感受到,光束密糖 Molasses,朱棣文(江苏太仓),A Single-Molecule Study of RNA Catalysis and Folding,Xiaowei Zhuang, Laura E. Bartley, Hazen P. Babcock, Rick Russell, Taekjip Ha, Daniel Herschlag, Steven Chu1Science, 2000,288,2048-2051,Howard Hughes Medical Inst

10、itute InvestigatorProfessor of Chemistry and Chemical BiologyProfessor of Physics,FRET time trace showing unfolding andrefolding of a ribozyme molecule,0.5s,intermediate,A Single-Molecule Study of protein Folding,Folding image of Single GCN-4 Dimers,Urea,Urea,Donor: R6G,Acceptor TxR,Single GCN-4 Dim

11、ersAt 0M Urea(pH6.1),Single GCN-4 DimersAt 7.4M Urea(pH 6.1),Strong AcceptorEfficient FRETFolded,Strong Donor &AcceptorLess Efficient FRETUnfolded,A Single-Molecule Study of gene expression in live cells,DNA,RNA,protein,Central dogma of molecular biology,Yu, Ji; Xiao, Jie; Ren, Xiaojia; Lao,Kaiqin;

12、Xie, X. SunneyScience, 311, 1600-1603 (2006),Stochastic protein expression in individual cells at the single molecule level,Cai, Long; Friedman, Nir; Xie, X. SunneyNature, 440, 358-362 (2006),Probing Gene Expression in Live Cells, One Protein Molecule at a Time,Tsr-Venus,Transcription of one mRNA by

13、 an RNA polymerase results from an infrequent and transient dissociation event of repressor from DNA,Scheme of live-cell observations of gene expression,Single-molecule detection of a fluorescent fusion protein, Tsr-Venus, in live E. coli cells.,Real-time monitoring of the expression of tsr-venus un

14、der the control of repressed lac promoter,Using Single-Molecule technology to Study the infectious pathway of virus,Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,Seisenberger G. et al. Science, 2001,294:1929-32,病毒：AAV 细胞：HeLa 荧光探剂：Cy5 (Carbocyanine dye), 1/2

15、5 仪器：CCD（电荷耦合探测器） 测量：74个细胞，1009 AAV- Cy5轨 迹，,病毒通过细胞表面时会慢下来。然后病毒接触细胞表面好几次，多数情况下没有应答，只有13%的病毒通过细胞表面,大约三分之一的病毒颗粒延规定好的途径定向移动,一旦其进入细胞，病毒有50%的几率达到细胞核,DNA Image taken by Evanescent Microscope (Single Molecule Observation),Low Background,By AFM,Progress in biophysics & Molecular biology 2002,OmpF porin imag

16、ed in buffer solution using AFM.,Molecule motor,分子马达，是对一大类广泛存在于细胞内部的能够把化学能（ATP）直接转换为机械能的蛋白分子的总称, 纳米机器,线性推进式马达分子自身能够沿着一条线性轨道作定向移动。如：微管上定向跑动的驱动蛋白一kinesin,旋转式其结构象马达一样，由一个定子与一个转子组成，依靠定子和转子之间的旋转运动来完成工作任务。最典型的旋转式分子马达是F1-ATP酶。它由两部分组成，一部分镶嵌在线粒体膜上，另一部分暴露在膜外。镶嵌在膜上的部分有三个亚基构成质子通过线粒体膜的通道，当质子流经这样的通道时产生力矩，从而推动暴露在膜

17、外的亚基的旋转，成为线粒体重要的运输工,Movie,Molecular Linear Motor Myosin,Molecular Linear Motor Myosin,References,Ch.Zander, J.Enderlein, R.A.Keller， Single Molecule Detection in SolutionXiaowei Zhuang, et al. A Single-Molecule Study of RNA Catalysis and Folding, Science, 2000,288,2048-2051 Seissenberger G. et al, Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus, Science, 2001,294:1929Cai, Long; Friedman, Nir; Xie, X. Sunney, Stochastic protein expression in individual cells at the single molecule level, Nature, 2006, 440, 358-362,