單分子生物物理學(xué)簡介Single molecule biophysics第三章單分子

1、23294Single moleculeAs of 2003 Single Molecules ceased publication and the articles are incorporated in ChemPhysChemlessfasterImpetus to single molecule Why we conduct single molecule or what single molecule can tell us?single molecules in diverse environments such as liquids, solids, membranes, sur

2、faces, and polymersSingle 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 chemistry, physics, and life sciences:methods of manipulating and modif

3、ying single particlessingle atoms and nanoparticles all kinds of ultrasensitive detection techniques such as spectroscopies and microscopies在單分子水平上對生物分子行為（包括在單分子水平上對生物分子行為（包括）的）的檢測以及在此基礎(chǔ)上的檢測以及在此基礎(chǔ)上的等，等，是分子生物是分子生物物理物理學(xué)的自然延伸和必然學(xué)的自然延伸和必然趨勢趨勢。生物單分子研究已成為。生物單分子研究已成為2121世紀生命世紀生命科學(xué)領(lǐng)域的一個重點研究方向，在生命科科學(xué)領(lǐng)域的一個重點研

4、究方向，在生命科學(xué)領(lǐng)域有著廣泛的應(yīng)用前景學(xué)領(lǐng)域有著廣泛的應(yīng)用前景 Why we conduct single molecule or what single molecule can tell us?ultrasensitivesensitivityThe 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

5、in the solvent, glass coverslips, and optical components, and from dark current in the detector. Genesis of noise?a small excitation volume, to reduce the backgroundhigh-efficiency collection opticsdetectors with high quantum efficiency and low dark noisecareful elimination of background fluorescenc

6、e: solvent, optics, detection method et al.Techniques to study the single moleculeLaser Scanning Confocal Microscopy (LSCM)Total Internal Reflection Fluorescence Microscopy (TIRFM)Near field Scanning Optical Microscopy (NSOM or SNOM)Scanning Probe Microscopy(SPM):Scanning Tunneling Microscope STMAto

7、m Force Microscope ( AFM )Optical tweezer/ optical trapOP or OTA 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在獲得諾貝爾獎的第二天，在獲得諾貝爾獎的

8、第二天，他騎著自行車，朝著目標往他騎著自行車，朝著目標往山路上攀爬，達到了目的地。山路上攀爬，達到了目的地。這種攀登高峰的踏實感受這種攀登高峰的踏實感受:也只有在努力過之后，才能也只有在努力過之后，才能真切地感受到真切地感受到 光束密糖光束密糖 Molasses朱棣文朱棣文(江蘇太倉江蘇太倉)A Single-Molecule Study of RNA Catalysis and Folding Xiaowei Zhuang, Laura E. Bartley, Hazen P. Babcock, Rick Russell, Taekjip Ha, Daniel Herschlag, Stev

9、en Chu1Science, 2000,288,2048-2051Howard Hughes Medical Institute InvestigatorProfessor of Chemistry and Chemical BiologyProfessor of Physics FRET time trace showing unfolding andrefolding of a ribozyme molecule0.5sintermediateA Single-Molecule Study of protein Folding Folding image of Single GCN-4

10、DimersUreaUreaDonor: R6GAcceptor TxRSingle GCN-4 DimersAt 0M Urea(pH6.1)Single GCN-4 DimersAt 7.4M Urea(pH 6.1)Strong AcceptorEfficient FRETFoldedStrong Donor &AcceptorLess Efficient FRETUnfoldedA Single-Molecule Study of gene expression in live cellsDNARNAproteinCentral dogma of molecular biolo

11、gy 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 levelCai, Long; Friedman, Nir; Xie, X. SunneyNature, 440, 358-362 (2006)Probing Gene Expression in Live Cells, One Protein Molecule at

12、a TimeTsr-Venus Transcription of one mRNA by an RNA polymerase results from an infrequent and transient dissociation event of repressor from DNA Scheme of live-cell observations of gene expressionSingle-molecule detection of a fluorescent fusion protein, Tsr-Venus, in live E. coli cells.Real-time mo

13、nitoring of the expression of tsr-venus under the control of repressed lac promoterUsing Single-Molecule technology to Study the infectious pathway of virusReal-time single-molecule imaging of the infection pathway of an adeno-associated virusSeisenberger G. et al. Science, 2001,294:1929-32 病毒：病毒：AA

14、V 細胞：細胞：HeLa 熒光探劑：熒光探劑：Cy5 (Carbocyanine dye), 1/25 儀器：儀器：CCD（電荷耦合探測器）（電荷耦合探測器） 測量：測量：74個細胞，個細胞，1009 AAV- Cy5軌軌 跡，跡， 病毒通過細胞表面時會慢病毒通過細胞表面時會慢下來。然后病毒接觸細胞下來。然后病毒接觸細胞表面好幾次，多數(shù)情況下表面好幾次，多數(shù)情況下沒有應(yīng)答，只有沒有應(yīng)答，只有13%的病的病毒通過細胞表面毒通過細胞表面 大約三分之一的病大約三分之一的病毒顆粒延規(guī)定好的毒顆粒延規(guī)定好的途徑定向移動途徑定向移動 一旦其進入細胞，一旦其進入細胞，病毒有病毒有50%的幾率的幾率達到細胞核

15、達到細胞核 DNA Image taken by Evanescent Microscope (Single Molecule Observation)Low BackgroundBy AFMProgress in biophysics & Molecular biology 2002OmpF porin imaged in buffer solution using AFM.Molecule motor分子馬達，是對一大類廣泛分子馬達，是對一大類廣泛存在于細胞內(nèi)部的能夠把存在于細胞內(nèi)部的能夠把化化學(xué)能（學(xué)能（ATP）直接轉(zhuǎn)換為直接轉(zhuǎn)換為機機械能械能的蛋白分子的總稱的蛋白分子的總稱 納

16、米機器納米機器線性推進式線性推進式馬達分子自身能夠沿著一條線性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)動軌道作定向移動。如：微管上定向跑動的驅(qū)動蛋白一蛋白一kinesin 旋轉(zhuǎn)式旋轉(zhuǎn)式其其結(jié)構(gòu)象馬達一樣，由一個定子與一個轉(zhuǎn)子組成，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(wù)。最典型的依靠定子和轉(zhuǎn)子之間的旋轉(zhuǎn)運動來完成工作任務(wù)。最典型的旋轉(zhuǎn)式分子馬達是旋轉(zhuǎn)式分子馬達是F1-ATP酶。它由兩部分組成，一部分鑲酶。它由兩部分組成，一部分鑲嵌在線粒體膜上，另一部分暴露在膜外。鑲嵌在膜上的部分嵌在線粒體膜上，另一部分暴露在膜外。鑲嵌在膜上

17、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為線粒體重要的運輸工 MovieReferencesCh.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