Single proteins at work

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Presentation transcript:

Single proteins at work

Enzyme reaction kinetics E•S k2 E0 + P E + S E0 k3 E

Enzyme reaction kinetics d[P] dt k2[E0][S] [S] + KM = = v = k2[E•S] KM= k-1 + k2 k1

Molecular Time scales 10-18 s 10-15 s 10-12 s 10-9 s 10-6 s 10-3 s 1 s second milli-second micro-second nano-second pico-second femto-second atto-second Talked about spatial scales, now time scales. What kind of motions on what time scale? Fs to a minute, minute to the age of universe.

Microsecond motion Small poly-peptide. Folding of proteins. Very complicated process, takes time. Simulation of 100 microseconds

The illusion of the ensemble

Enzymes are over a million times smaller than a honey bee! The challenge of one Enzymes are over a million times smaller than a honey bee!

Neher & Sakmann, Nobel prize medicine 1991 Ion channels Early single protein measurements (1970’s) Neher & Sakmann, Nobel prize medicine 1991

Early optical attempts absorption

Focus on one fluorescence

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

Ribozyme

Ribozyme

Fluctuations: ET model Flavin:NADH oxidoreductase (Fre) Fluorescence decay rate:

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

Correlation

New tools waiting time probability density of  mean waiting time for Michaelis-Menten kinetics waiting time correlation function intensity correlation function

Cholesterol Oxidase

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

Correlation of on-times

-galactosidase -galactosidase

English et al., Nat. Chem. Biol. 2, 87 (2006) 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 learned Enzymes fluctuate on a broad range of time scales Reaction kinetics are dispersed, only the average is measured in ensembles