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PDBsum entry 2gjh
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De novo protein
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PDB id
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2gjh
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References listed in PDB file
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Key reference
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Title
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Mis-Translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution.
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Authors
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G.Dantas,
A.L.Watters,
B.M.Lunde,
Z.M.Eletr,
N.G.Isern,
T.Roseman,
J.Lipfert,
S.Doniach,
M.Tompa,
B.Kuhlman,
B.L.Stoddard,
G.Varani,
D.Baker.
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Ref.
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J Mol Biol, 2006,
362,
1004-1024.
[DOI no: ]
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PubMed id
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Abstract
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We recently used computational protein design to create an extremely stable,
globular protein, Top7, with a sequence and fold not observed previously in
nature. Since Top7 was created in the absence of genetic selection, it provides
a rare opportunity to investigate aspects of the cellular protein production and
surveillance machinery that are subject to natural selection. Here we show that
a portion of the Top7 protein corresponding to the final 49 C-terminal residues
is efficiently mis-translated and accumulates at high levels in Escherichia
coli. We used circular dichroism, size-exclusion chromatography, small-angle
X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show
that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely
stable, homo-dimeric structure. Based on the solution structure, we engineered
an even more stable variant of CFr by disulfide-induced covalent circularisation
that should be an excellent platform for design of novel functions. The
accumulation of high levels of CFr exposes the high error rate of the protein
translation machinery. The rarity of correspondingly stable fragments in natural
proteins coupled with the observation that high quality ribosome binding sites
are found to occur within E. coli protein-coding regions significantly less
often than expected by random chance implies a stringent evolutionary pressure
against protein sub-fragments that can independently fold into stable
structures. The symmetric self-association between two identical mis-translated
CFr sub-domains to generate an extremely stable structure parallels a mechanism
for natural protein-fold evolution by modular recombination of protein
sub-structures.
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Figure 7.
Figure 7. Comparison of the Top7 and CFr structures. (a)
and (b) Ribbon diagrams of residues 3–51 from one subunit of
the CFr NMR structure (green) superimposed on the corresponding
region of the Top7 X-ray structure (purple). The backbone RMSD
value over these residues is 1.12 Å. The two diagrams are
related by a 90° rotation around the vertical axis in the
plane of the page.
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Figure 9.
Figure 9. Backbone Dynamics of CFr. (a) ^15N T[1]
measurements; (b) ^15N T[2] measurements; (c) ^15N HetNOE
measurements.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
362,
1004-1024)
copyright 2006.
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