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In Vitro Single Molecules Single molecule enzymology

In Vitro Single Molecules Single molecule enzymology. Yonil Jung. Ever fluctuating single enzyme molecules: Michaselis-Menten equation revisited. Nonspecifically bound proteins spin while diffusing along DNA. Mass action at the single-molecule level. Single molecule studies.

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In Vitro Single Molecules Single molecule enzymology

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  1. In Vitro Single Molecules Single molecule enzymology Yonil Jung Ever fluctuating single enzyme molecules: Michaselis-Menten equation revisited Nonspecifically bound proteins spin while diffusing along DNA Mass action at the single-molecule level

  2. Single molecule studies

  3. Key papers in single-molecule enzymology Alex E. Knight, Single enzyme studies: A historical perspective, 2011

  4. Experimental scheme BP English et al. Nature Chemical Biology 2, 87 ‐ 94 (2006)

  5. Turnover time traces

  6. Michaelis-Menten equation with dynamic disorder Mono-exponential Multi-exponential 10 mM 20 mM 50 mM 100 mM

  7. Mono-exponential Multi-exponential

  8. Memory effects of a single enzyme molecule

  9. 2D joint probability distribution

  10. Non-specific DNA-protein interaction PC Blainey et al. Nature Structural & molecular Biology 16, 1224 ‐ 1229 (2009)

  11. Experimental Scheme

  12. Diffusion constants 1. One dimensional diffusion k R D = 2. Rotation-coupled translational diffusion k R3 D = F(e)

  13. Effect of protein size on diffusion constants

  14. Rotation-coupled translation

  15. Analysis of diverse set of DNA-binding proteins

  16. Small energy barriers in helical sliding

  17. Interactions between two molecules by single molecule study Typical in vitro single molecule studies: few picomolar to nanomolar

  18. Decreasing detection volume MJ Levene et al. Science 299, 682 ‐ 686 (2003)

  19. Increasing local concentration by confinement JJ Ben ́ıtez et al. Methods in enzymology 472, 41 ‐ 60 (2010)

  20. Reversibly encapsulating molecules by nanofabricated “dimples” Min JuShon and Adam E. Cohen. J. Am. Chem. Soc. 134 (35), 14618–14623 (2012)

  21. Molecular affinity probed by joint occupancy

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