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