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PDBsum entry 252l
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References listed in PDB file
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Key reference
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Title
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Generation of ligand binding sites in t4 lysozyme by deficiency-Creating substitutions.
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Authors
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E.Baldwin,
W.A.Baase,
X.J.Zhang,
V.Feher,
B.W.Matthews.
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Ref.
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J Mol Biol, 1998,
277,
467-485.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
perfect match.
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Abstract
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Several variants of T4 lysozyme have been identified that sequester small
organic ligands in cavities or clefts. To evaluate potential binding sites for
non-polar molecules, we screened a number of hydrophobic large-to-small mutants
for stabilization in the presence of benzene. In addition to Leu99-->Ala,
binding was indicated for at least five other mutants. Variants Met102-->Ala
and Leu133-->Gly, and a crevice mutant, Phe104-->Ala, were further
characterized using X-ray crystallography and thermal denaturation. As predicted
from the shape of the cavity in the benzene complex, mutant Leu133-->Gly also
bound p-xylene. We attempted to enlarge the cavity of the Met102-->Ala mutant
into a deep crevice through an additional substitution, but the double mutant
failed to bind ligands because an adjacent helix rearranged into a non-helical
structure, apparently due to the loss of packing interactions. In general, the
protein structure contracted slightly to reduce the volume of the void created
by truncating substitutions and expanded upon binding the non-polar ligand, with
shifts similar to those resulting from the mutations.A polar molecule binding
site was also created by truncating Arg95 to alanine. This creates a highly
complementary buried polar environment that can be utilized as a specific
"receptor" for a guanidinium ion. Our results suggest that creating a deficiency
through truncating mutations of buried residues generates "binding potential"
for ligands with characteristics similar to the deleted side-chain. Analysis of
complex and apo crystal structures of binding and non-binding mutants suggests
that ligand size and shape as well as protein flexibility and complementarity
are all determinants of binding. Binding at non-polar sites is governed by
hydrophobicity and steric interactions and is relatively permissive. Binding at
a polar site is more restrictive and requires extensive complementarity between
the ligand and the site.
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Figure 7.
Figure 7. Changes in solvent structure near residue 104.
Solvents are denoted by starred atoms and the broken lines
connect solvent atom pairs that are within hydrogen bonding
distance. (a) WT* lysozyme; (b) mutant F104A; (c) benzene
complex F104A/BZ.
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Figure 10.
Figure 10. (a) Superposition of M102A/M106A (filled bonds)
on WT* (open bonds) showing the "alternative" conformation with
hydrogen bonds indicated by broken lines. (b) Superposition of
residues 106 to 114 of the "alternative" conformation of
M102A/M106A (filled atoms and bonds), together with the
"wild-type-like" conformation of M102A/M106A (thin open bonds
and starred atoms), on WT* (open thick bonds and atoms), based
on atoms 81 to 161.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1998,
277,
467-485)
copyright 1998.
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Secondary reference #1
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Title
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A cavity-Containing mutant of t4 lysozyme is stabilized by buried benzene.
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Authors
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A.E.Eriksson,
W.A.Baase,
J.A.Wozniak,
B.W.Matthews.
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Ref.
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Nature, 1992,
355,
371-373.
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PubMed id
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Secondary reference #2
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Title
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Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance.
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Authors
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D.C.Muchmore,
L.P.Mcintosh,
C.B.Russell,
D.E.Anderson,
F.W.Dahlquist.
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Ref.
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Methods Enzymol, 1989,
177,
44-73.
[DOI no: ]
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PubMed id
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Secondary reference #3
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Title
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Structure of bacteriophage t4 lysozyme refined at 1.7 a resolution.
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Authors
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L.H.Weaver,
B.W.Matthews.
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Ref.
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J Mol Biol, 1987,
193,
189-199.
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PubMed id
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