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
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
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
Mono-exponential
Multi-exponential
10 mM
20 mM
50 mM
100
mM
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
D=
k
R
2. Rotation-coupled translational diffusion
D=
k
F(e)
3
R
Effect of protein size on diffusion
constants
Rotation-coupled translation
Analysis of diverse set of DNAbinding 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)
Increasing local concentration by
confinement
JJ Ben ́ıtez et al. Methods in enzymology 472, 41 ‐ 60 (2010)
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