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 10  M 20  M 50  M 100  M Mono-exponential Multi-exponential

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 2. Rotation-coupled translational diffusion k R D = kR3kR3 F 

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

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

21. Molecular affinity probed by joint occupancy