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

Igor Demonstration of analysis with models of polymer elasticity

Reverse Engineering of the giant muscle protein titin

The elastic protein titin is the third filament of muscle

Electron micrographs of isolated titin molecules

Machina Carnis

Titin: a complex mechanical protein B C D Adapted from Linke, 2007, Cardiovascular Research (in press)

Measuring the extensibility of titin in a single isolated cardiac fiber

Elasticity of PEVK

Electron micrographs of PEVK_I27 polyprotein

Persistence length of PEVK

Elasticity of N2B

V11P V15P V13P wt Y9P

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

Mechanical design of the extracellular matrix: fibronectin

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

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

Fluorescently labeled fibronectin assembled by CHO cells

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

Mechanical design of the extracellular matrix: polysaccharides

Polysaccharides cellulose amylose

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

Periodate oxidation cleaves the rings of pectin

Ubiquitin chains form a mechanical signalling system in cells

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

a0=4 x 10-4; Dx=0.25

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