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PDBsum entry 1vkt
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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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Mapping the functional surface of insulin by design: structure and function of a novel a-Chain analogue.
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Authors
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Q.X.Hua,
S.Q.Hu,
B.H.Frank,
W.Jia,
Y.C.Chu,
S.H.Wang,
G.T.Burke,
P.G.Katsoyannis,
M.A.Weiss.
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Ref.
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J Mol Biol, 1996,
264,
390-403.
[DOI no: ]
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PubMed id
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Abstract
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Functional surfaces of a protein are often mapped by combination of X-ray
crystallography and mutagenesis. Such studies of insulin have yielded
paradoxical results, suggesting that the native state is inactive and
reorganizes on receptor binding. Of particular interest is the N-terminal
alpha-helix of the A-chain. Does this segment function as an alpha-helix or
reorganize as recently proposed in a prohormone-convertase complex? To correlate
structure and function, we describe a mapping strategy based on protein design.
The solution structure of an engineered monomer ([AspB10, LysB28, ProB29]-human
insulin) is determined at neutral pH as a template for synthesis of a novel
A-chain analogue. Designed by analogy to a protein-folding intermediate, the
analogue lacks the A6-A11 disulphide bridge; the cysteine residues are replaced
by serine. Its solution structure is remarkable for segmental unfolding of the
N-terminal A-chain alpha-helix (A1 to A8) in an otherwise native subdomain. The
structure demonstrates that the overall orientation of the A and B chains is
consistent with reorganization of the A-chain's N-terminal segment.
Nevertheless, the analogue's low biological activity suggests that this segment,
a site of clinical mutation causing diabetes mellitus, functions as a preformed
recognition alpha-helix.
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Figure 2.
Figure 2. A, Ribbon model of T6 insulin hexamer in 2-Zn crystal form (Protein Data Bank identifier 2ZN; Baker et al.,
1988). Protomers are shown in red and green. The central Zn-binding sites are coordinated by HisB10 (white). The
view is along the 3-fold symmetry axis of the hexamer. B, Surface representation of T-state protomer (2-Zn molecule 1)
showing residues HisB10, ProB28 and LysB29 (green; sites of mutation in DKP-insulin) and cystine A6--A11 (yellow;
sites of serine substitution in DKP-[A6-A11]
Ser
). Because the latter is inaccessible, the yellow surface is not well seen.
The view is rotated from that shown in A to visualize most clearly the relevant protein surfaces. C, Stereo depiction
of internal environment of A6--A11 disulphide bridge (yellow) in 2-Zn molecule 1; neighbouring aliphatic side-chains
are shown in red (A-chain) and blue (B-chain) as indicated.
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Figure 5.
Figure 5. Differences in
1
H-NMR chemical shifts
between DKP-insulin and DKP-[A6-A11]
Ser
at neutral pH
are shown by residue: a, amide resonances, b, a
resonances, c, b methylene resonances, and d, other
side-chain resonances. For each residue only the
difference largest in magnitude is shown. A-chain
residues are numbered 1 to 21; B-chain residues, 22 to 51.
Arrows indicate sites of serine substitution in DKP-[A6-
A11]
Ser
.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1996,
264,
390-403)
copyright 1996.
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