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Single proteins at work

Single proteins at work. k 1. k 2. k 3. E•S. E 0 + P. k -1. E 0. E. Enzyme reaction kinetics. E + S. k 2 [E 0 ][S]. =. [S] + K M. d[P]. k -1 + k 2. dt. K M =. k 1. Enzyme reaction kinetics. = v. = k 2 [E•S]. atto-second. femto-second. pico-second. nano-second.

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Single proteins at work

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  1. Single proteins at work

  2. k1 k2 k3 E•S E0 + P k-1 E0 E Enzyme reaction kinetics E + S

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

  4. atto-second femto-second pico-second nano-second micro-second milli-second second Molecular Time scales 10-18 s 10-15 s 10-12 s 10-9 s 10-6 s 10-3 s 1 s

  5. Microsecond motion

  6. The illusion of the ensemble

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

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

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

  16. Correlation

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

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

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