第三章單分子生物物理學

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 biophys

2、ics?,Why we conduct single molecule or what single molecule can tell us?,How to study the single molecules?,,single molecules in diverse environments such as liquids, solids, membranes, surfaces, and poly

3、mers,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 single molecule investigations in chemi

4、stry, 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 sin

5、gle molecule biophysics?,在單分子水平上對生物分子行為（包括構(gòu)象變化、相互作用、相互識別等）的實時﹑動態(tài)檢測以及在此基礎上的操縱﹑調(diào)控等，是分子生物物理學的自然延伸和必然趨勢。生物單分子研究已成為21世紀生命科學領域的一個重點研究方向，在生命科學領域有著廣泛的應用前景,Why we conduct single molecule or what single molecule can tell us?,In co

6、ntrast with ensemble methods, single molecule experiments provide information on distributions and time trajectories of observables that would otherwise be hidden,single molecule methods,Sensitive or ultrasensitive,,sens

7、itivity,,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 coverslips, an

8、d 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 noi

9、se,careful elimination of background fluorescence: solvent, optics, detection method et al.,How to increase S/N?,Techniques to study the single molecule,Laser Scanning Confocal Microscopy (LSCM),Total Internal Reflectio

10、n Fluorescence Microscopy (TIRFM),Near field Scanning Optical Microscopy (NSOM or SNOM),Scanning Probe Microscopy(SPM):,Scanning Tunneling Microscope STM,Atom Force Microscope ( AFM ),Optical tweezer/ optical trapO

11、P or OT,,A Single-Molecule Study of RNA Catalysis and Folding,The Nobel Prize in Physics 1997"for development of methods to cool and trap atoms with laser light",Steven ChuUSA,Claude Cohen-TannoudjiFrance,W

12、illiam D. Phillips USA,在獲得諾貝爾獎的第二天，他騎著自行車，朝著目標往山路上攀爬，達到了目的地。這種攀登高峰的踏實感受:也只有在努力過之后，才能真切地感受到,光束密糖 ? Molasses,朱棣文(江蘇太倉),A Single-Molecule Study of RNA Catalysis and Folding,Xiaowei Zhuang, Laura E. Bartley, Hazen P. Bab

13、cock, Rick Russell, Taekjip Ha, Daniel Herschlag, Steven Chu1Science, 2000,288,2048-2051,,Howard Hughes Medical Institute InvestigatorProfessor of Chemistry and Chemical BiologyProfessor of Physics,FRET time trace sho

14、wing 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 DimersAt 0M Urea

15、(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,,,,

16、Central dogma of molecular biology,Yu, Ji; Xiao, Jie; Ren, Xiaojia; Lao,Kaiqin; Xie, X. SunneyScience, 311, 1600-1603 (2006),Stochastic protein expression in individual cells at the single molecule level,Cai, Long; Fr

17、iedman, 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 an RNA polymerase results from an infrequent and trans

18、ient 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

19、 expression of tsr-venus under 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-as

20、sociated virus,Seisenberger G. et al. Science, 2001,294:1929-32,病毒：AAV 細胞：HeLa 熒光探劑：Cy5 (Carbocyanine dye), 1/25 儀器：CCD（電荷耦合探測器） 測量：74個細胞，1009 AAV- Cy5軌 跡，,病毒通過細胞表面時會慢下來。然后病毒接觸細胞表面好幾次，多數(shù)情況下沒有應答，只有13%的病毒通過細胞表面,大約三分之一

21、的病毒顆粒延規(guī)定好的途徑定向移動,一旦其進入細胞，病毒有50%的幾率達到細胞核,DNA Image taken by Evanescent Microscope (Single Molecule Observation),Low Background,,By AFM,Progress in biophysics & Molecular biology 2002,OmpF porin imaged in buffer solu

22、tion using AFM.,Molecule motor,分子馬達，是對一大類廣泛存在于細胞內(nèi)部的能夠把化學能（ATP）直接轉(zhuǎn)換為機械能的蛋白分子的總稱,? 納米機器,線性推進式?馬達分子自身能夠沿著一條線性軌道作定向移動。如：微管上定向跑動的驅(qū)動蛋白一kinesin,旋轉(zhuǎn)式?其結(jié)構(gòu)象馬達一樣，由一個定子與一個轉(zhuǎn)子組成，依靠定子和轉(zhuǎn)子之間的旋轉(zhuǎn)運動來完成工作任務。最典型的旋轉(zhuǎn)式分子馬達是F1-ATP酶。它由兩部分組成，一部分鑲嵌在

23、線粒體膜上，另一部分暴露在膜外。鑲嵌在膜上的部分有三個亞基構(gòu)成質(zhì)子通過線粒體膜的通道，當質(zhì)子流經(jīng)這樣的通道時產(chǎn)生力矩，從而推動暴露在膜外的亞基的旋轉(zhuǎn)，成為線粒體重要的運輸工,Movie,Molecular Linear Motor ? Myosin,,Molecular Linear Motor ? Myosin,References,Ch.Zander, J.Enderlein, R.A.Keller， Single Molecu

24、le 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