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PDBsum entry 2b7x
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References listed in PDB file
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Key reference
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Title
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Sequential reorganization of beta-Sheet topology by insertion of a single strand.
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Authors
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M.Sagermann,
W.A.Baase,
B.W.Matthews.
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Ref.
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Protein Sci, 2006,
15,
1085-1092.
[DOI no: ]
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PubMed id
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Abstract
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Insertions, duplications, and deletions of sequence segments are thought to be
major evolutionary mechanisms that increase the structural and functional
diversity of proteins. Alternative splicing, for example, is an intracellular
editing mechanism that is thought to generate isoforms for 30%-50% of all human
genes. Whereas the inserted sequences usually display only minor structural
rearrangements at the insertion site, recent observations indicate that they may
also cause more dramatic structural displacements of adjacent structures. In the
present study we test how artificially inserted sequences change the structure
of the beta-sheet region in T4 lysozyme. Copies of two different beta-strands
were inserted into two different loops of the beta-sheet, and the structures
were determined. Not surprisingly, one insert "loops out" at its
insertion site and forms a new small beta-hairpin structure. Unexpectedly,
however, the second insertion leads to displacement of adjacent strands and a
sequential reorganization of the beta-sheet topology. Even though the insertions
were performed at two different sites, looping out occurred at the C-terminal
end of the same beta-strand. Reasons as to why a non-native sequence would be
recruited to replace that which occurs in the native protein are discussed. Our
results illustrate how sequence insertions can facilitate protein evolution
through both local and nonlocal changes in structure.
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Figure 1.
Schematic representation of the [beta]-sheet of T4 lysozyme.
The sheet structure consists of Strands I, II, and III and turns
T-1, T-2, and T-3. The sequences to be inserted are shown in
red. In mutant L30c, the inserted amino acid sequence
corresponds to Strand II plus Turn T-2 and is inserted after
Tyr24. In mutant L31d, the inserted sequence corresponds to Turn
T-2 plus Strand III and is inserted after residue Leu33. The
color-coding shown here is maintained in all figures.
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Figure 2.
Illustration of some possible folds of the [beta]-sheet
domain as a result of the insertion L30c. The inserted sequence
(red) contains Strand II and Turn T-2. (A) In the simplest
scenario, the inserted structure loops out at the insertion
site, i.e., at Turn T-1. The neighboring structure retains the
native conformation, leaving the identical parent sequence
(yellow) unchanged. (B) In a second scenario, the insert
sequence displaces the identical parent sequence, forcing it to
loop out at Turn T-2. (C) In yet another scenario (the one that
is observed), the sequence that is displaced in B continues so
as to displace the sequence in Strand III. The structure will
then be forced to loop out at T-3. In going from scenario B to
C, the wild-type turn structure T-2 (Gly-Ile-Gly) is restored.
In contrast, however, Strand II now replaces Strand III, causing
substitutions in this region.
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The above figures are
reprinted
from an Open Access publication published by the Protein Society:
Protein Sci
(2006,
15,
1085-1092)
copyright 2006.
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Secondary reference #1
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Title
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Long distance conformation changes in a protein engineered by modulated sequence duplication
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Authors
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M sagermann,
L.Gay,
B.W.Mattews.
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Ref.
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proc natl acad sci usa, 2003,
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9191.
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Secondary reference #2
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Title
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Structural characterization of an engineered tandem repeat contrasts the importance of context and sequence in protein folding.
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Authors
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M.Sagermann,
W.A.Baase,
B.W.Matthews.
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Ref.
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Proc Natl Acad Sci U S A, 1999,
96,
6078-6083.
[DOI no: ]
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PubMed id
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Figure 2.
Fig. 2. (A) Initial electron density showing the overall
conformation of the duplicated sequence, as seen in space group
P3[2]21. The WT* structure, omitting residues 36-42 (shown as a
ribbon drawing) was subject to 10 cycles of rigid-body
refinement in the mutant lysozyme cell. The calculated phases
and structure factors, F[c], were used to calculate a map with
amplitudes (F[mutant]  F[c]) at
3.0-Å resolution. The density in the vicinity of the
deleted residues, contoured at 2.5  , is shown.
(B) Electron density after refinement of the inserted region in
space group P3[2]21. Coefficients are (2F[o]-F[c]). The
structure factors, F[c], and phases were calculated from the
refined model including the inserted region. The resolution is
2.5 Å, and the map is contoured at 1.0 . (C)
Superposition of the overall structure of the duplication mutant
in space group P3[2]21 (blue bonds) on WT* lysozyme (green
bonds). The inserted region in the mutant structure is
highlighted in yellow.
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Figure 3.
Fig. 3. (A) Map showing the initial electron density for
the inserted region of molecule A in space group P2[1].
Amplitudes are (2F[o]-F[c]) weighted by REFMAC (15) where the
structure factors, F[c], and phases were calculated from the
refined model including the inserted region. The map was
calculated at 2.5-Å resolution and contoured at 1.0 . The
density in the vicinity of residues 40i-43i is not well defined
and could not be fit by a well-defined model. (B) Electron
density for molecule B of crystal form P2[1]. This map was
calculated with the same coefficients, contouring, and
resolution as in A. (C) Superposition of the C  trace of
the two copies of mutant L20 in crystal form P2[1] (molecule A,
blue; molecule B, mauve) and wild-type T4 lysozyme (green). The
sequence of the insert is highlighted in yellow for molecule A
and in orange for molecule B. The structural rearrangements of
loop 18-25 in molecule B are clearly visible. The superpositions
were based on the -carbon
atoms of residues 51-80 within the amino-terminal domain.
Because of slight changes in the hinge-bending angle the
C-terminal domains appear out of register although the
respective structures within these regions are very similar.
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