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PDBsum entry 1sif
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Structural protein
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PDB id
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1sif
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin.
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Authors
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C.G.Benítez-Cardoza,
K.Stott,
M.Hirshberg,
H.M.Went,
D.N.Woolfson,
S.E.Jackson.
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Ref.
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Biochemistry, 2004,
43,
5195-5203.
[DOI no: ]
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PubMed id
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Abstract
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The stability, dynamic, and structural properties of ubiquitin and two multiple
hydrophobic core mutants were studied. One of the mutants (U4) has seven
substitutions in the hydrophobic core (M1L, I3L, V5I, I13F, L15V, V17M, and
V26L). On average, its side chains are larger than the wild-type, and it can
thus be thought of as having an overpacked core. The other mutant (U7) has two
substitutions (I3V and I13V). On average, it has smaller side chains than the
wild-type, and it can therefore be considered to be underpacked. The three
proteins are well-folded and show similar backbone dynamics (T(1), T(2), and
HNOE values), indicating that the regular secondary structure extends over the
same residue ranges. The crystallographic structure of U4 was determined. The
final R(factor) and R(free) are 0.198 and 0.248, respectively, at 2.18 A
resolution. The structure of U4 is very similar to wild-type ubiquitin.
Remarkably, there are almost no changes in the positions of the C(alpha) atoms
along the entire backbone, and the hydrogen-bonding network is maintained. The
mutations of the hydrophobic core are accommodated by small movements of side
chains in the core of mutated and nonmutated residues. Unfolding and refolding
kinetic studies revealed that U4 unfolds with the highest rates; however, its
refolding rate constants are very similar to those of the wild-type protein.
Conversely, U7 seems to be the most destabilized protein; its refolding rate
constant is smaller than the other two proteins. This was confirmed by
stopped-flow techniques and by H/D exchange methodologies. This work illustrates
the possibility of repacking the hydrophobic core of small proteins and has
important implications in the de novo design of stable proteins.
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