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Figure 3.
Figure 3. Hydrophobic packing involving residues in the CD loop
of the cis SH2 structure provides stabilization energy. a,
Overlay of 20 lowest energy structures including the CD loop,
the central  -sheet
and  A
of the cis (left) and trans (right) conformers. Side chains of
Leu 254 and Pro 287 are yellow. His 291 and Glu 250 are also
labeled. b, Overlay of 20 lowest energy structures (rotated
clockwise with respect to (a)) showing the CD loop of the cis
(left) and trans (right) conformers. Side chains of Ile 282, Ala
281 and Cys 288 are labeled and shown in yellow. Additional side
chains are included without labels for clarity. c,
Three-dimensional 13C-edited NOESY experiment showing
through-space proximity between the  -methyl
protons of Ile 282 and one of the -methylene
protons of Cys 288. The NOE is observed only for the cis
conformer (left panel). The total number of NOEs unique to the
cis and trans structures is shown in Table 1. d,
Three-dimensional 15N-edited TOCSY experiment illustrating the
nondegenerate resonance frequencies for the Cys 288 -methylenes
in the cis conformer (left). The same protons resonate at a
single frequency in the trans conformer (right). e, Expansion of
the 1H-15N HSQC spectra showing the amide signal of Gly 260
(6260) in the cis and trans forms. Left, unmodified, reduced Itk
SH2 domain. Middle, spectrum acquired following reaction of the
Itk SH2 domain with glutathione disulfide (GSSG) (20 mM GSSG, pH
7.4, 40 min, 25 °C). Right, spectrum acquired following reaction
with methyl methane thiosulfonate (MMTS) (5 mM MMTS, pH 7.4, 20
min). The percentage of SH2 domain in the cis conformation in
each of these experiments as measured by peak volumes of Gly 260
(cis) and Gly 260 (trans) was 48, 5 and 32% for the reduced,
GSSG-treated and MMTS-treated proteins, respectively. The
backbone amide resonance of Cys 288 was monitored over the
course of both reactions and the reactions were allowed to
proceed until no further chemical shift changes occurred. The
completeness of the S-glutathiolation reaction was also assessed
by separation of the domain with nondenaturing isoelectric
focusing (IEF) gel electrophoresis over a pH range of 3.5 -10 as
described^33.
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