In Vitro Single Molecules Single molecule enzymology Yonil Jung Ever fluctuating single enzyme molecules: Michaselis-Menten equation revisited Nonspecifically.

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Presentation transcript:

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

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

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

Turnover time traces

Michaelis-Menten equation with dynamic disorder 10  M 20  M 50  M 100  M Mono-exponential Multi-exponential

Mono-exponential Multi-exponential

Memory effects of a single enzyme molecule

2D joint probability distribution

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

Experimental Scheme

Diffusion constants 1. One dimensional diffusion 2. Rotation-coupled translational diffusion k R D = kR3kR3 F 

Effect of protein size on diffusion constants

Rotation-coupled translation

Analysis of diverse set of DNA- binding proteins

Small energy barriers in helical sliding

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

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

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

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

Molecular affinity probed by joint occupancy