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PDBsum entry 1kda
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Hydrolase (phosphoric diester)
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PDB id
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1kda
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References listed in PDB file
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Key reference
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Title
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Stabilization of a strained protein loop conformation through protein engineering.
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Authors
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A.Hodel,
R.A.Kautz,
R.O.Fox.
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Ref.
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Protein Sci, 1995,
4,
484-495.
[DOI no: ]
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PubMed id
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Abstract
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Staphylococcal nuclease is found in two folded conformations that differ in the
isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different
conformations of the residue 112-117 loop. The cis form is favored over the
trans with an occupancy of 90%. Previous mutagenesis studies have shown that
when Lys 116 is replaced by glycine, a trans conformation is stabilized relative
to the cis conformation by the release of steric strain in the trans form.
However, when Lys 116 is replaced with alanine, the resulting variant protein is
identical to the wild-type protein in its structure and in the dominance of the
cis configuration. The results of these studies suggested that any nuclease
variant with a non-glycine residue at position 116 should also favor the cis
form because of steric requirements of the beta-carbon at this position. In this
report, we present a structural analysis of four nuclease variants with
substitutions at position 116. Two variants, K116E and K116M, follow the
"beta-carbon" hypothesis by favoring the cis form. Furthermore, the crystal
structure of K116E is nearly identical to that of the wild-type protein. Two
additional variants, K116D and K116N, provide exceptions to this simple
"beta-carbon" rule in that the trans conformation is stabilized relative to the
cis configuration by these substitutions. Crystallographic data indicate that
this stabilization is effected through the addition of tertiary interactions
between the side chain of position 116 with the surrounding protein and water
structure. The detailed trans conformation of the K116D variant appears to be
similar to the trans conformation observed in the K116G variant, suggesting that
these two mutations stabilize the same conformation but through different
mechanisms.
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Figure 7.
Fig. 7. A: Hydrogen bondingpattern between residues 1 I5 nd 118 of nuclease A (Hynes & Fox, 1991) andthesurrounding
ordered water. Residues 115-1 8 adopt a type IVa &turn with a hydrogen bond between the carbonyl oxygen of Tyr 115 and
thebackboneamide of Asn 118. B: Hydrogen bonding pattern between residues 115 and 18 of 116D and thesurrounding
ordered water. The Asp 116 side chain replaces thebackbone of Tyr 115 n many of the hydrogen bonds observed in the wild-
typestructure. C: omparison of the residue 112-118 loopconformationsfromthe K116D variant (white) nd the 116G
nuclease variant (Hodel et al., 1992) (green). Oxygen atoms re colored red; nitrogen is colored blue.
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Figure 8.
Fig. 8. Electrondensitysurroundingresidues112-1 18 of the K116Nvariant.Shownisthe F, - F SA-omit mapcalculatedusing
thenucleaseAstructureexcluding the atoms of residues112-1 18. Mapis contouredat 1.5~. Densityyieldsthe conformation
of esidues112-114and116-1 17, ut conformation of residue115isnotclear. Two odels areshownwiththe map. A model
similar to hat of K116Dwasbiltandrefined to the data. B Amodelbased on he conformation found in the K116Gvariant
is also consistentwiththeelectron density. Although thedensity in the SA-omitmap favors the conformation of 115in
the K116G model (B), stericconsiderationssuggestthattheK116D (A) is morelikely to be adopted by K116N.
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The above figures are
reprinted
by permission from the Protein Society:
Protein Sci
(1995,
4,
484-495)
copyright 1995.
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