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PDBsum entry 2f2q
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References listed in PDB file
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Key reference
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Title
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Guanidinium derivatives bind preferentially and trigger long-Distance conformational changes in an engineered t4 lysozyme.
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Authors
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M.S.Yousef,
N.Bischoff,
C.M.Dyer,
W.A.Baase,
B.W.Matthews.
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Ref.
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Protein Sci, 2006,
15,
853-861.
[DOI no: ]
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PubMed id
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Abstract
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The binding of guanidinium ion has been shown to promote a large-scale
translation of a tandemly duplicated helix in an engineered mutant of T4
lysozyme. The guanidinium ion acts as a surrogate for the guanidino group of an
arginine side chain. Here we determine whether methyl- and ethylguanidinium
provide better mimics. The results show that addition of the hydrophobic
moieties to the ligand enhances the binding affinity concomitant with reduction
in ligand solubility. Crystallographic analysis confirms that binding of the
alternative ligands to the engineered site still drives the large-scale
conformational change. Thermal analysis and NMR data show, in comparison to
guanidinium, an increase in protein stability and in ligand affinity. This is
presumably due to the successive increase in hydrophobicity in going from
guanidinium to ethylguanidinium. A fluorescence-based optical method was
developed to sense the ligand-triggered helix translation in solution. The
results are a first step in the de novo design of a molecular switch that is not
related to the normal function of the protein.
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Figure 2.
Stereo pair showing that the backbone structures of L20/R63A
lysozyme complexed with guanidinium ion (red), methylguanidinium
(yellow), and ethylguanidinium (green) are virtually identical.
The structure of L20/R63A in the absence of ligand (cyan)
differs substantially.
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Figure 3.
Details of the stabilizing interactions of the loop at the C
terminus of the engineered helix in the mutant L20/R63A in the
presence of guanidinium (A), methylguanidinium (B), and
ethylguanidi-nium (C). The superimposed F[o]-F[c] difference
maps contoured at 3[sigma] (red) define the position of the
ligands. The resolution of the maps is 1.45 A, 1.7 A, and 1.8 A,
respectively. The methylated and ethylated ligands adopt
alternative conformations as shown. (D) Interactions made by
Arg63 in the lysozyme (Molecule B, PDB code 262L). Similar
interactions are made by Arg52 in wild-type lysozyme.
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The above figures are
reprinted
from an Open Access publication published by the Protein Society:
Protein Sci
(2006,
15,
853-861)
copyright 2006.
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Secondary reference #1
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Title
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Use of sequence duplication to engineer a ligand-Triggered, Long-Distance molecular switch in t4 lysozyme.
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Authors
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M.S.Yousef,
W.A.Baase,
B.W.Matthews.
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Ref.
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Proc Natl Acad Sci U S A, 2004,
101,
11583-11586.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Details of the interactions that stabilize the loop
at the C terminus of the duplicated helix. (a) L20 (the design
template). (b) L20/R63A in the presence of guanidinium.
Distances (black) are shown in Å; in green are the
corresponding distances in the WT structure. The superimposed
F[o] - F[c] difference map contoured at 3.3  (red) defines the
position of the ligand.
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Figure 2.
Fig. 2. (a) Superposition of liganded (red) on the
unliganded (cyan) forms of L20/R63A. As representative examples,
the alternative positions of Ser-44 are labeled. On the lower
left and right are simulated-annealing omit maps (contoured at
1.1 ) with backbone
representations of the helix extended in both directions. (b)
Detailed sketch showing the structures of the liganded (Upper)
and the unliganded (Lower) forms. The "inserted" residues
(Asn-40-Ile-50) are colored orange, and the "parent" residues
(Asn-51-Ile-61, renumbered because of the 11-residue insert) are
colored blue. The vertical bars connecting the two structures
show the location of helix B in WT. In the presence of the
guanidinium ion (Upper), the inserted helix (in orange) extends
at its N terminus. In the absence of the ion (Lower), the
inserted sequence occupies the position of helix B and the
parent sequence extends the helix at its C terminus.
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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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Secondary reference #3
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Title
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Long-Distance conformational changes in a protein engineered by modulated sequence duplication.
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Authors
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M.Sagermann,
L.Gay,
B.W.Matthews.
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Ref.
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Proc Natl Acad Sci U S A, 2003,
100,
9191-9195.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. (A) Backbone representation of the structure of T4
lysozyme mutant L20 in which residues 39-50 are duplicated in
tandem (17). The original residues 39-50 (yellow) have a
conformation similar to helix B of WT. The duplicated residues
(red) extend helix B at its N terminus. Residues 51-56 are
green. Their conformation is the same as WT and helps stabilize
the conformation at the C terminus of helix B. A and Fig. 2 were
created with PYMOL (Warren DeLano, DeLano Scientific, San
Carlos, CA). (B) The details of the conformation of the
N-terminal extension of helix B as seen in the structure of L20
(A). Most of the contacts within the extension are hydrophobic
in nature and, for clarity, only the side chains that contribute
to these interactions are shown. The color-coding of the
backbone is the same as in C. [B and C and Figs. 3 and 4 were
made with MOLSCRIPT and RASTER3D (27).] (C) Conformation at the
N terminus of helix B in WT (compare B). As in B, only those
side chains that make contacts within the loop are shown.
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Figure 3.
Fig. 3. Superposition of L20pg on L20. The two tandem
repeat sequences are yellow and red, respectively. The
corresponding C-terminal loop residues 51-56 are green and dark
green. As representative examples, the alternative positions of
residues 42 and 52 are labeled.
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