Force-spectroscopy

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Force-spectroscopy of single proteins
II mechanical engineering in biological systems
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Igor Demonstration of analysis with models of
polymer elasticity
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Reverse Engineering of the giant muscle protein
titin
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The elastic protein titin is the third filament
of muscle
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Electron micrographs of isolated titin molecules
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Machina Carnis
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Titin a complex mechanical protein
A
B
C
D
Adapted from Linke, 2007, Cardiovascular Research
(in press)
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Measuring the extensibility of titin in a single
isolated cardiac fiber
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Elasticity of PEVK
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Electron micrographs of PEVK_I27 polyprotein
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Persistence length of PEVK
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Elasticity of N2B
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V11P
V15P
V13P
wt
Y9P
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Understand the mechanical design of titin in
humans
Understand the molecular design of its modules
Create titin phenotypes in mice
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Mechanical design of the extracellular
matrixfibronectin
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A complex web of proteins and polysaccharides
that provides the mechanical scaffold for organs
and tissues
ECM
cell membrane
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Fibronectin a major, cell binding component of
the ECM
NMR structure of 10F3. The RGD residues are
identified in the picture.
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Fluorescently labeled fibronectin assembled by
CHO cells
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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
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Mechanical design of the extracellular
matrixpolysaccharides
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Polysaccharides
cellulose
amylose
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If we mechanically stretch a sugar ring, it gets
longer by switching from a chair to a boat
conformation
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Periodate oxidation cleaves the rings of pectin
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Ubiquitin chains form a mechanical
signallingsystem in cells
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From Weissman, Nature Reviews, 2001, 2169-178
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a04 x 10-4 Dx0.25
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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