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Figure 3.
Figure 3. Representative solution structures of: a,
(GluB16, GlyB24, desB30)-insulin at pH 8.0; and b,
HisB16-insulin (PDB code 1HLS) at pH 2.4 in aqueous solution
[Ludvigsen et al 1994] together with the corresponding surface
representations (c and d). The helical stretches (in a and b)
are shown in blue, and selected side-chains are annotated. In b,
the side-chains of B24, B25 and B26 are arranged as is observed
in crystal structures of wild-type insulin. In a, PheB25 has
moved into the position usually occupied by PheB24. This
structural change is quantitatively characterized by a value of
0.42(±0.17) Å for the mean displacement of the C^α
atom of PheB25 between the average and ensemble structures of
the (GluB16, GlyB24, desB30)-mutant. Furthermore, when the
helical regions (A2 to A9, A14 to A20 and B9 to B19) are used to
align the ensemble of structures for each mutant, a displacement
of only 1 Å is obtained between the average positions of
the PheB25 C^α atom of the (GluB16, GlyB24, desB30)-insulin and
PheB24 C^α atom of HisB16-insulin. Accordingly, the average
values of torsion angles, χ^1 and χ^2, are
50.3(±4.2)° and −96.8(±5.6)°,
respectively, for the PheB25 residue in the present mutant as
compared to values of 53.5(±5.3)° and
−79.2(±4.0)°, respectively, for the equivalent
PheB24 residue in the HisB16 mutant structure. TyrB26 remains
approximately in its original position. Overall, this change
perturbs the turn following the central B-chain helix and bends
the usual extended structure of the C-terminal strand. The
surface shown in d is rotated a little around the vertical axis
compared to b to display the side-chains of B24 and B26 which
are clearly differently exposed as compared to c. Side-chains of
interest and importance for receptor binding are color-coded and
annotated with sequence position in c and d. In a and c the bend
of the C-terminal residues and the accompanying reorientation
result in the exposure of a different surface compared to b and
d, i.e. in particular ValA3 and TyrB26 become more exposed.
Mutant insulins were constructed by oligonucleotide-directed
mutagenesis, fermented in yeast, and purified as described
[Markussen et al 1987 and Brange et al 1988]. Mutants are
expressed as single-chain miniproinsulin precursors,
PheB1...LysB29-Ala-Ala-Lys-GlyA1...AsnA21. The connecting
peptide is cleaved off using lysyl endopeptidase (Achromobacter
protease I, EC 3.4.21.50; Wako Inc., Osaka, Japan). The removal
of ThrB30 has no effect on the biological potency. Figures of
molecules were produced using Insight (Molecular Simulations
Inc.) (c and d) and MOLSCRIPT [Kraulis 1991] combined with
RASTER3D [Bacon and Anderson 1988 and Merritt and Murphy 1994]
(a and b).
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