Single Molecule Force Spectroscopy

Transcription

1 Single Molecule Force Spectroscopy force pn rupture force counts cantilever-position x rupture force [pn] E.-L. Florin, V. T. Moy, H. E. Gaub, Science 264, 415 (1994) 1

2 Streptavidin/Biotin Unbinding Simulation H. Grubmüller, B. Heymann, P. Tavan, Science 271, 997, 1996 M. Rief, H. Grubmüller, Physikalische Blätter, Feb. 2001, p

3 Streptavidin/Biotin Unbinding Simulation H. Grubmüller, B. Heymann, P. Tavan, Science 271, 997, 1996 M. Rief, H. Grubmüller, Physikalische Blätter, Feb. 2001, p

4 Streptavidin/Biotin Unbinding Simulation 3

5 Mechanoenzymatics: Titin: Force buffers and Force Sensors 4

6 Titin is a giant muscle protein 3000 kda, ca. 400g of body weight 5

7 Titin is a giant muscle protein 3000 kda, ca. 400g of body weight 6

8 Titin consists of many Ig domains and provides muscle elasticity N-terminus I-band A-band C-terminus Ig domain actin myosin 7

9 Force probe experiments (AFM, opt. tweezer): --> Ig domains as shock absorbers Gautel et al., Science 1997, Biophysical Journal 1999 Linke and Fernandez, J. of Muscle and Cell Mot

10 Enforced unfolding of the titin Ig27 domain unfolding force [pn] spontaneous unfolding simulation [1] M. Rief et al., Science 276 (1997) 1109 [2] M. Carrion-Vazquez et al., PNAS 96 (1999) 3694 loading rate [pn/s] H. Wagner, H. Grubmüller, Europ.Biophys.J. 29 (2000) 273 (abstract) 9

11 Why is there a titin kinase? N-terminus I-band A-band C-terminus titin kinase actin myosin 10

12 Titin kinase structure: a force sensor? catalytic loop regulatory tail terminal beta sheets 2Å resolution Gautel et al., Nature 395 (1998)

13 Sample force probe MD simulation: asymmetric rupture of beta sheets v = 4 A/ns, 22 ns total 12

14 Unfolding forces and rupture events N C C N Predicted unfolding force: pn AFM exp. (Gaub et al.): 70 pn N C F. Gräter, J. Shen, H. Jiang, M. Gautel, H. Grubmüller, Biophys. J. (2005) 13

15 Parallel vs. sequential rupture N C F. Gräter, J. Shen, H. Jiang, M. Gautel, H. Grubmüller, Biophys. J. (2005) 14

16 Asymmetric rupture of the terminal beta sheets N-terminus N C C-terminus 15

17 Ruptures of the terminal beta sheets parallel rupture: high force resistance sequential rupture: low force resistance F. Gräter, J. Shen, H. Jiang, M. Gautel, H. Grubmüller, Biophys. J. (2005) 16

18 AFM experiments reveal ATP fingerprint E. Puchner et al., PNAS 105, (2008) 17

19 MD: Complete Titin Kinase unfolding v = 0.4 m/s, 500 ns total 18

20 Exposure of binding site -- with ATP v = 0.4 m/s, 500 ns total 19

21 Simulation: Complete Titin Kinase unfolding with ATP no ATP E. Puchner et al., PNAS 105, (2008) 20

22 Force Histograms quantify ATP peak E. Puchner et al., PNAS 105, (2008) 21

23 Unbinding forces depend on time scale due to activated barrier crossing: F(x): applied force G(x) + V(x,t) G(x): energy landscape of unperturbed system V(x,t): spring potential p(x): reaction coordinate probability distribution 22

24 Same process, 10 3 times slower: F(x): applied force G(x) + V(x,t) G(x): energy landscape of unperturbed system V(x,t): spring potential p(x): reaction coordinate probability distribution 23

25 Same process, 10 3 times slower: F(x): applied force G(x) + V(x,t) G(x): energy landscape of unperturbed system V(x,t): spring potential p(x): reaction coordinate probability distribution 24

26 Experiments on Crook s Fluctuation Theorem D. Collin et al, Nature 437, (2005) 25

27 DNA / Zinc Finger Binding Specificity Small differences in binding free energy determine specificity Target for protein engineering eventual therapeu;c applica;on of zinc finger nucleases 26

28 DNA / Zinc Finger Binding Specificity λ=0 a c DNA- Protein Complex λ=0.5 ΔΔG bind Water λ=1 b Nucleo;de X Nucleo;de Y Free energy changes calculated along physically impossible but computationally realisable alchemical transformation pathways 27

29 DNA / Zinc Finger Binding Specificity 28

30 DNA / Zinc Finger Binding Specificity 29

31 DNA / Zinc Finger Binding Specificity Consistent results with two independent methods (equilibrium and non- equilibrium sampling) Excellent agreement with measured specificity 30