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PDBsum entry 2cw1
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De novo protein
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PDB id
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2cw1
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References listed in PDB file
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Key reference
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Title
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Design of lambda cro fold: solution structure of a monomeric variant of the de novo protein.
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Authors
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Y.Isogai,
Y.Ito,
T.Ikeya,
Y.Shiro,
M.Ota.
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Ref.
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J Mol Biol, 2005,
354,
801-814.
[DOI no: ]
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PubMed id
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Abstract
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One of the classical DNA-binding proteins, bacteriophage lambda Cro, forms a
homodimer with a unique fold of alpha-helices and beta-sheets. We have
computationally designed an artificial sequence of 60 amino acid residues to
stabilize the backbone tertiary structure of the lambda Cro dimer by simulated
annealing using knowledge-based structure-sequence compatibility functions. The
designed amino acid sequence has 25% identity with that of natural lambda Cro
and preserves Phe58, which is important for formation of the stably folded
structure of lambda Cro. The designed dimer protein and its monomeric variant,
which was redesigned by the insertion of a beta-hairpin sequence at the
C-terminal region to prevent dimerization, were synthesized and biochemically
characterized to be well folded. The designed protein was monomeric under a wide
range of protein concentrations and its solution structure was determined by NMR
spectroscopy. The solved structure is similar to that of a monomeric variant of
natural lambda Cro with a root-mean-square deviation of the polypeptide
backbones at 2.1A and has a well-packed protein core. Thus, our knowledge-based
functions provide approximate but essential relationships between amino acid
sequences and protein structures, and are useful for finding novel sequences
that are foldable into a given target structure.
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Figure 4.
Figure 4. NMR analyses of solution structure of designed
Cro monomer. (a) 1H-15N HSQC spectrum of SN4m. (b) Short-range
NOE connectivity. The left notations of aH, NN and ab indicate
Ha-HN, HN-HN and Hb-HN, respectively. (c) b-Structural topology.
Only the unambiguous NOE connectivity between the backbone CaH
and NH protons on separate b-strands are indicated by arrows.
Hydrogen bonds included in the structure calculations are
indicated by broken lines.
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Figure 6.
Figure 6. Solution structures of designed Cro monomer. (a)
Stereo representation of the ten energy-minimized NMR structures
of SN4m. Each of the ten backbone traces is superimposed on the
average structure to minimize the RMSD of the backbone atoms
(0.63 Å) and displayed with the hydrophobic residue
side-chains (Leu5, Leu7, Phe10, Val11, Leu23, Leu25, Leu29,
Ile30, Val33, Leu34, Leu38, Val40, Val42, Ile50, Val52, Ile54,
Ile59, and Phe63) in pink bars. (b) Comparison between the
average solution structure of SN4m and the X-ray
crystallographic structure of the natural l Cro monomer (1orc)
in stereo representation. The average NMR structure (red) is
superimposed on the natural Cro monomer structure (white) with
the minimal backbone RMSD of 2.1 Å.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2005,
354,
801-814)
copyright 2005.
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