Single proteins at work

Enzyme reaction kinetics

E + Sk1

k-1

E•Sk2 E0 + P

E0k3 E

Enzyme reaction kinetics

= k2[E•S]d[P]dt = v

k2[E0][S]

[S] + KM

=

KM=k-1 + k2

k1

Molecular Time scales

secondmilli-second

micro-second

nano-second

pico-second

femto-second

atto-second

1 s10-3 s10-6 s10-9 s10-12 s10-15 s10-18 s

Microsecond motion

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The illusion of the ensemble

The challenge of one

Enzymes are over a million times smaller than a honey bee!

Ion channels

Neher & Sakmann, Nobel prize medicine 1991

Early single protein measurements (1970’s)

Early optical attemptsabsorption

Focus on onefluorescence

Ribozyme

X. Zhang et al., Science 296, 1473 (2002)

Ribozyme

Ribozyme

Fluctuations: ET model

Flavin:NADH oxidoreductase (Fre)

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k(t) = k0e−βx( t )Fluorescence decay rate:

Lifetime

H. Yang et al., Science 302, 262 (2003)

Correlation

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C(t) = Δk(0)−1Δk(t)−1

New tools

waiting time

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τ =

probability density of τ

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f (τ ) =

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τ = τf (τ )0

∞

∫ dτ mean waiting time

for Michaelis-Menten kinetics

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1

τ=

k2[S]

[S]+KM

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Cτ (m) =Δτ (0)Δτ (m)

Δτ 2waiting time correlation function

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CI (t) =ΔI(0)ΔI(t)

ΔI2intensity correlation function

Cholesterol Oxidase

Single molecule turnovers

H.P. Lu et al., Science 282, 1877 (2002)

Correlation of on-times

-galactosidase

-galactosidase

Single molecule assay

English et al., Nat. Chem. Biol. 2, 87 (2006)

Reaction trajectories

Concentration dependence

Conformer interconversion

Comparison with MM kinetics

Intensity correlation

fluctuation of k2

New lessons learnedEnzymes fluctuate on a broad range of time scales

Reaction kinetics are dispersed, only the average is measured in ensembles