L12 - Biophysical Chemistry Chemistry 24a Winter Term...

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Biophysical Chemistry Chemistry 24a Winter Term 2009-10 Instructor: Sunney I. Chan Lecture 12 February 10, 2010 Single-Molecule Methods. Principles and Applications of Single-Molecule Methods in the Study of Biological Macromolecules 3 Introduction “If an alien species would make an ensemble-averaged observation of the human population on Earth, they would conclude that each person has one testicle and one ovary!” -- Steven Chu, Nobel laureate for Physics ,1997 Limitations of ensemble method: static vs. dynamic heterogeneity Power of SM approach: manipulate single molecules and observe dynamic processes Basic principles: spatial and temporal distribution of parameters rather than average values Three challenges: ¾ Surface immobilization: streptavidin-biotin interaction ¾ Detection of single molecules: fluorescence or force ¾ Improving sensitivity: increasing signal-noise ratio SM FRET Principles of SM spectroscopy Optical tweezers AFM SM Michaelis-Menten Equation Verification of Jarzynski’s Equality Techniques Applications Ribozyme folding and catalysis 4 Brief Timeline of SM Spectroscopy 1986 Taekjip Ha (UIUC) First detected FRET between 2 single molecules Calvin Quate (Stanford) Gerd Binnig (IBM Lab, Zürich) Invented the atomic force microscope capable of manipulating single molecules Christof Gerber (U. Basel) Arthur Ashkin (Bell Labs) Invented the optical tweezers to trap molecules and atoms 1996 Steven Chu (Berkeley) Carlos Bustamante (Berkeley) Measured mechanical properties of single DNA molecules by force spectroscopy 1992 Sunney Xie (Harvard) Hermann Gaub (Technische Universität München) 1999 2006 2003 Xiaowei Zhuang (Harvard) Detected infection pathway of single influenza viruses Mattias Rief (Technische Universität München) Steven Block (Stanford) Directly observed single base-pair stepping of RNA polymerase during transcription 2005 Observed expression of single protein molecules in live cells by FRET Determined force needed to break a glycosidic bond using AFM
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Why single molecule (SM) experiments? SM experiment vs experiment on a bulk sample In bulk experiment, one measures ensemble average over N molecules (N a large number approaching N A ). In SM experiment, one is making observations on a single molecule at a given location over time. So, one is monitoring molecular fluctuations and sampling the dynamics of the molecule. (Can average over a number of molecules in space). Ergodic hypothesis : Ensemble average = Time average! Transcription mediated by RNA polymerase II population distribution
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How to achieve SM experiments? The following slides courtesy of Hung-wen Li, Department of Chemistry, NTU
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light microscope video camera Dig-bio dsDNA = DI-Biotin double-labeled double-stranded DNA (DNA labeled with digoxigenin-dUTP and biotin ) Application Streptavidin-biotin interaction Streptavidin is a 52,800 dalton[1] tetrameric protein purified from the bacterium Streptomyces avidinii . It finds wide use in molecular biology through its extraordinarily strong affinity for biotin (also known as vitamin
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L12 - Biophysical Chemistry Chemistry 24a Winter Term...

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