Force-spectroscopyof single proteins

II: mechanical engineering in biological systems

Igor Demonstration of analysis with models of

polymer elasticity

Reverse Engineering of Reverse Engineering of the the

giant muscle protein titingiant muscle protein titin

The elastic protein titin is the third filament of muscle

Electron micrographs of isolated titin molecules

Machina Carnis

A

B

C

D

Titin: a complex mechanical protein

Adapted from Linke, 2007, Cardiovascular Research (in press)

Measuring the extensibilityof titin in a single

isolated cardiac fiber

Elasticity of PEVK

Electron micrographs of PEVK_I27 polyprotein

Persistence length of PEVK

Elasticity of N2B

V13P

V11P V15P

wt Y9P

Understand the mechanical design of titin in humans

Create titin phenotypes in mice

Understand the molecular design of its modules

Mechanical design of Mechanical design of the extracellular matrix:the extracellular matrix:

fibronectinfibronectin

A complex web of proteins and polysaccharides that provides

the mechanical scaffold for organs and tissues

cell membrane

ECM

NMR structure of 10F3. The RGD residues are identified in the picture.

Fibronectin: a major, cell binding component of the ECM

Fluorescently labeled fibronectin assembled by CHO cells

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

Mechanical design of Mechanical design of the extracellular matrix:the extracellular matrix:

polysaccharidespolysaccharides

amylose

Polysaccharidescellulose

If we mechanically stretch a sugar ring, it gets longer by switching from a chair to a boat conformation

0.45 nm0.55 nm

Periodate oxidation cleaves the rings of pectin

Ubiquitin chains form a Ubiquitin chains form a mechanical signallingmechanical signalling

system in cellssystem in cells

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

0=4 x 10-4; x=0.25

1.0

0.8

0.6

0.4

0.2

0.0

Pu

nfo

ld (

fo

r t=

1/0

.05

min

-1 )

12 3 4 5 6 7 8 9

102 3

n

n=4

F = 57 pN

n

nkT

Fdx

etentP

01),(

A)

B)

F

n

proteasome polyubiquitin

Targeted protein

Conclusions

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.

1.- Single molecule force spectroscopy combined with protein engineering can examine the mechanical design of complex protein structures

4.- This paradigm can be extended to many other biological systems