1. Single proteins at work

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

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

4. 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.

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

6. The illusion of the ensemble

7. 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!

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

9. Early optical attempts
absorption

10. Focus on one
fluorescence

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

12. Ribozyme

13. Ribozyme

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

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

16. Correlation

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

18. Cholesterol Oxidase

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

20. Correlation of on-times

21. -galactosidase
-galactosidase

22. English et al., Nat. Chem. Biol. 2, 87 (2006)
Single molecule assay
English et al., Nat. Chem. Biol. 2, 87 (2006)

23. Reaction trajectories

24. Concentration dependence

25. Conformer interconversion

26. Comparison with MM kinetics

27. Intensity correlation
fluctuation of k2

28. New lessons learned Enzymes fluctuate on a broad range of time scales
Reaction kinetics are dispersed, only the average is measured in ensembles