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PDBsum entry 2ck2
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Signaling protein
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PDB id
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2ck2
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References listed in PDB file
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Key reference
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Title
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Designing an extracellular matrix protein with enhanced mechanical stability.
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Authors
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S.P.Ng,
K.S.Billings,
T.Ohashi,
M.D.Allen,
R.B.Best,
L.G.Randles,
H.P.Erickson,
J.Clarke.
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Ref.
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Proc Natl Acad Sci U S A, 2007,
104,
9633-9637.
[DOI no: ]
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PubMed id
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Abstract
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The extracellular matrix proteins tenascin and fibronectin experience
significant mechanical forces in vivo. Both contain a number of tandem repeating
homologous fibronectin type III (fnIII) domains, and atomic force microscopy
experiments have demonstrated that the mechanical strength of these domains can
vary significantly. Previous work has shown that mutations in the core of an
fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that
domain significantly: The composition of the core is apparently crucial to the
mechanical stability of these proteins. Based on these results, we have used
rational redesign to increase the mechanical stability of the 10th fnIII domain
of human fibronectin, FNfn10, which is directly involved in integrin binding.
The hydrophobic core of FNfn10 was replaced with that of the homologous,
mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc
retains both the three-dimensional structure and the cell adhesion activity of
FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of
the engineered protein FNoTNc increase by approximately 20% to match those of
TNfn3. Thus, we have specifically designed a protein with increased mechanical
stability. Our results demonstrate that core engineering can be used to change
the mechanical strength of proteins while retaining functional surface
interactions.
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Figure 2.
Fig. 2. FNoTNc retains the structure of its parents. (a)
Stereo view showing an overlay of the backbone traces of FNoTNc
(green), FNfn10 (blue), and TNfn3 (red). The only regions where
FNfn10 and FNoTNc differ significantly are in the C–C' and
F–G loops, both regions known to be flexible in FNfn10 (7).
(b) Stereo view showing an overlay of FNoTNc (green) and TNfn3
(red). The core residues have the same conformation.
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Figure 3.
Fig. 3. Unfolding forces. (a) Unfolding forces of TNfn3
(blue), FNoTNc (red), and FNfn10 (black). FNoTNc unfolds at the
same force as TNfn3. Only FNfn10 shows double peaks, indicting
the presence of an unfolding intermediate. These traces were
collected at a retraction speed of 1,000 nm/s. (b) Histograms of
unfolding forces of TNfn3 (blue), FNoTNc (red), and FNfn10
(black) at a retraction speed of 1,000 nm/s. The modal unfolding
forces are 127 ± 3 pN (n = 305), 125 ± 3 pN (n =
332), and 104 ± 5 pN (n = 172), respectively [see also
supporting information (SI) Fig. 5].
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