1. II: mechanical engineering
Force-spectroscopy
of single proteins
II: mechanical engineering
in biological systems

2. Igor Demonstration of analysis with models of polymer elasticity

3. Reverse Engineering of the giant muscle protein titin

5. The elastic protein titin is the third filament of muscle

6. Electron micrographs of isolated titin molecules

9. Machina Carnis

10. Titin: a complex mechanical proteinB C D
Adapted from Linke, 2007, Cardiovascular Research (in press)

11. Measuring the extensibility of titin in a single
isolated cardiac fiber

17. Elasticity of PEVK

18. Electron micrographs of PEVK_I27 polyprotein

19. Persistence length of PEVK

20. Elasticity of N2B

26. V11P
V15P
V13P wt
Y9P

27. Understand the mechanical design of titin in humans
Understand the molecular design of its modules
Create titin phenotypes in mice

28. Mechanical design of the extracellular matrix: fibronectin

29. A complex web of proteins and polysaccharides that provides
the mechanical scaffold for organs and tissues
ECM
cell membrane

30. Fibronectin: a major, cell binding component of the ECM
NMR structure of 10F3. The RGD residues are identified in the picture.

32. Fluorescently labeled fibronectin assembled by CHO cells

38. Mechanical hierarchies define the triggers of cellular activity
Mechanical unfolding of protein domains helps to keep the cells mechanically bonded.
Mechanical hierarchies define the triggers of cellular activity
Cell
binding
cryptic
binding
cryptic
binding

39. Mechanical design of the extracellular matrix: polysaccharides

40. Polysaccharides
cellulose
amylose

41. If we mechanically stretch a sugar ring, it gets longer
by switching from a chair to a boat conformation
0.55 nm
0.45 nm

43. Periodate oxidation cleaves the rings of pectin

46. Ubiquitin chains form a mechanical signalling system in cells

48. From Weissman, Nature Reviews, 2001, 2:169-178

52. a0=4 x 10-4; Dx=0.25

56. Conclusions 1.- Single molecule force spectroscopy combined with
protein engineering can examine the mechanical design
of complex protein structures
2.- Titin has a complex mechanical design with multiple
mechanical elements that combine to create the finely tuned
muscle elasticity.
3.- The extensibility of titin can be calculated from
single molecule data and then scaled up
to explain elasticity in situ.
4.- This paradigm can be extended to many other biological systems