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PDBsum entry 1bnf
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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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Disulfide mutants of barnase. I: changes in stability and structure assessed by biophysical methods and X-Ray crystallography.
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Authors
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J.Clarke,
K.Henrick,
A.R.Fersht.
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Ref.
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J Mol Biol, 1995,
253,
493-504.
[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
percentage match of
95%.
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Abstract
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In this series of papers, we examine the effects of introducing disulfide bonds
on the properties, structure and thermodynamics of a small globular protein,
barnase. Three mutants have been made, in each of which a single crosslink
confers different properties. Two of the disulfide bonds, between residues 43
and 80 (43-80) and between residues 85 and 102 (85-102), stabilise the protein,
relative to both wild-type and the corresponding (reduced) dithiol forms: 85-102
is more stable than predicted from the entropic destabilisation of the unfolded
state; 43-80 is less stable than predicted. The third disulfide bond, between
residues 70 and 92 (70-92) destabilises the protein relative to both wild-type
and the corresponding dithiol form, implying significant disruption of the
folded protein on formation of the disulfide bond. Crystal structures of the
three mutant proteins have been solved. All three proteins have essentially the
same fold as wild-type, but with left-handed disulfide bonds, which have
dihedral geometries that have not been observed in naturally occurring
disulfides. In the very stable mutant 85-102, there is no significant difference
between the mutant and wild-type structures: these data do not explain the large
stability of this protein. The disulfide bond at 43-80 induces small structural
rearrangements close to the site of the disulfide bond, associated with some
local disorder: the crosslink appears to decrease the stability of the native
form of the protein. The destabilising disulfide bond at 70-92 induces
considerable structural change, with displacement of a loop and consequent
disruption of a stabilising salt-bridge. Our studies do not support the view
that the conformation of the disulfide bond is crucial in determining the
stability of the mutant proteins.
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Figure 1.
Figure 1. General view of wild-type barnase showing
the sites of introduction of disulfide bonds between
residues 43 and 80 (43--80), 70 and 92 (70--92) and 85
and 102 (85--102). Picture prepared using the program
MOLSCRIPT (Kraulis, 1991).
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Figure 7.
Figure 7. The site of the disulfide bond of 70--92.
A, Superposition of chain C of 70--92 on chain C of wild-
type (red). There are two different conformations of
mutant observed, type I (yellow) and type II (green)
(Table 5). These differ in the conformation of the disulfide
bond. B, Superposition of chain C of 70--92, type I only
(yellow), on chain C of wild-type (red), some side-
chain N atoms are shown in blue. The salt-bridge between
Arg 69 and Asp 93 in wild-type is disrupted in the mutant.
In the mutant crystal structure the side-chain is solvated
and incompletely defined. In the mutant the side-chain of
Lys66 occupies the cavity left by Arg69, but no hydrogen
bonds are formed with Asp93.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1995,
253,
493-504)
copyright 1995.
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