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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. - PowerPoint PPT Presentation
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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