in vitro single molecules single molecule enzymology yonil jung ever fluctuating single enzyme...
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In Vitro Single MoleculesSingle 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
Year Paper Enzyme
1961 First measurement of the catalytic activity of single enzyme molecules
b-Galactosidase
1995 First demonstration of static heterogeneity in native enzyme
molecules
LDH
1995 Visualization of single substrate turnovers
Myosin
1998 Measurement of dynamic heterogeneity and observation of “memory” effects
Cholesterol oxidase
2006 Reconciliation of single molecule and Michaelis-Menten kinetics
b-Galactosidase
Experimental scheme
BP English et al. Nature Chemical Biology 2, 87 ‐ 94 (2006)
Turnover time traces
Michaelis-Menten equation with dynamic disorder
10 mM
20 mM
50 mM
100 mM
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 =
D =
kR3
F( )e
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
Interactions Dissociation constants
Strong protein-protein interaction few picomolar
Weak protein-protein interaction few micromolar to millimolar
Biotin-avidin femtomolar
22mer oligonucleotides few nanomolar
Typical in vitro single molecule studies: few picomolar to nanomolar
Decreasing detection volume
MJ Levene et al. Science 299, 682 ‐ 686 (2003)
JJ Ben Gıtez 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