Force-spectroscopy of single proteins

1. Force-spectroscopy of single proteins I: Basic elements of protein mechanics

2. Are proteins exposed to mechanical forces in-vivo? extracellular matrix connector complex P0 fibronectin Schwann cell Cytoskeleton: actin, etc. tenascin cell fascilin Cadherin NCAM Basal Lamina: tenascin fibronectin etc. Anchor proteins Dystrophin Cytoskeleton titin synapse cell-matrix muscle F ion channels

3. Mechanical forces are a natural protein denaturant. Denaturant = Force (physiological) Proteins unfold and extend under a stretching force reaction coordinate (Dx; end-to-end length) If we then relax the force, the protein folds!

4. We can stretch a single protein and measure how does the restoring force changes with the extension. mirrors laser Photodiode (Force) cantilever protein linear actuator (extension) .

6. Modified VEECO detector head Physik Instrumente “pico cube” actuator

8. Electron micrographs of isolated titin molecules

9. Polypeptides are freely jointed segments with complex chemical groups branching out on the sides.

10. Increased entropy makes polymers look crooked and pushes them to collapse

11. d Path of a particle undergoing Brownian motion

12. A polymer looks like the path of a particle in Brownian motion l

13. If we had a 1 meter long polypeptide…………. 1 meter it would spontaneously collapse into .

14.  x6 x3 x4 x7 x5 x2 x1 x F l1 l2 l3 F l6 l7 li l5 l4 The Freely Jointed Chain (FJC) model of entropic elasticity

15. Proteins are folded amino acid polymers

16. Ten thousand water molecules at room temperature Switch to and activate VMD cluster program

17. Folded proteins have a bonded structure that helps them resist the constant bombardment caused by Brownian motion. Small protein ubiquitin cartoon representation

18. The bonds that hold the protein together can be though of as an energy barrier that has to be overcome to unravel the protein.

19. AFM ready proteinengineering

21. DNA engineering

22. Protein purification

23. AFM ready polyproteins!

24. Force-extension relationship of a polyprotein

28. Igor Demonstration of analysis with models of polymer elasticity

29. Much of what we see can be explained with a simple model(for now)

30. How often does Brownian motion overcomes the energy barrier and causes the protein to unfold spontaneously ? Conformational change E kT end-to-end length t0 ~ 10-12 seconds

31. How long do we have to wait for Brownian motion to overcome an energy barrier ? pico seconds 1067years seconds Biology Too strong Brownian death E =

32. Control in Biology is accomplished by reducing energy barriers. Then, unlikely events will occur over short time periods DE

37. After mechanical unfoldinga protein can refold without errors (mostly)

41. Misfolding traps the linker between I27 modules

42. With all these measurements wecompile a rough energy landscape

44. How much does a single amino acid contributes to the contour length ?(or what is li for a protein)

45. 1.51 Å 1.33 Å 1.46 Å Lcontour= 4.30 Å The end-to-end length of an amino acid vs its contour length Lend-to-end= 3.6 Å

47. DLc= 2 nm

48. Controlling Chain Length

50. Varying the location of the disulfide bonds across the I27 structure allows us to measure the contour length of a single amino acid