Biomedical Engineering Reference
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in protein ROA spectra. In addition, these spectra also contain many bands
from the loops and turns located between secondary structural motifs that
are important for determining tertiary structure plus discrete bands from
some side chains that can serve as reporters of protein secondary and ter-
tiary structure. The sensitivity of protein ROA spectra to these details of
structure is apparent in these spectra. For example, the ROA spectra of hen
lysozyme are dominated by two positive bands in the amide III region at
∼
1300 and 1340 cm
−
1
both arising from
α
-helix, but with these two bands
1340 cm
−
1
band dis-
appears immediately when the protein, or peptide or virus, is dissolved in
D
2
O. This indicates that N-deformations make a significant contribution to
the generation of this ROA band as the corresponding N-D deformations con-
tribute to normal modes several hundred wave numbers lower; and that, in
proteins and viruses, the corresponding sequences are exposed to solvent and
not buried in hydrophobic regions where amide protons can take far longer
to exchange. Although the positive
reporting on different aspects of
α
-helix. The positive
∼
1300 cm
−
1
also
changes in D
2
O, again suggesting some contribution from N-H deformations,
in proteins containing a large amount of
α
-helix ROA band at
∼
-helix in a protected hydrophobic
environment much of its intensity is often retained. The positive
α
∼
1300 and
1340 cm
−
1
ROA bands have, therefore, been assigned to non-hydrated and
hydrated forms of
-helix, respectively [3, 36].
This interpretation is supported by studies of water molecules in high-
resolution protein X-ray crystal structures [37-40]. For example, Blundell
et al. [37] found that water induces distortions and curvature in
α
α
-helices, with
59
◦
,
44
◦
with
n
=3
.
55 residues per
average Ramachandran
φ
,
ψ
angles of
−
−
66
◦
,
41
◦
turn for
α
-helices in hydrophobic environments, and
φ
,
ψ
angles of
−
−
with
n
=3
.
62 for
-helices in hydrophilic environments. Sundaralingam and
Sekharudu [40] later identified three types of hydrogen bonding interactions
between water molecules and the peptide backbone. In
external
hydration, the
C=O
i
group already engaged in intrachain helix hydrogen bonding to NH
i
+4
forms a hydrogen bond with an external water molecule. In
three centred
hy-
dration, the amide group forms a hydrogen bond with the carbonyl group as
well as water molecules and hence participates in a bifurcated or three-centred
hydrogen bond. In
water inserted
hydration, the water molecule inserts itself
between the C=O
i
and NH
i
+4
groups, replacing the
i
α
-helix hydro-
gen bond with a hydrogen-bonded bridge. Such water inserted hydration of
α
→
i
+4
α
-helices in protein crystal structures gives rise to a range of conformations
including 3
10
-helical elements (equivalent to type III turns) and type I turns
containing
i
→
i
+ 3 hydrogen bonds stabilized by the inserted water molecule
and extended
-strands. Since these hydrated structures occupy contiguous
regions of the Ramachandran surface, such hydrated structures may represent
intermediates in the helix-coil transition pathway and so provide insight into
the role of the aqueous medium in promoting helix (un)folding [40].
External backbone hydration is more common than the other two types
and may be especially relevant to aqueous solution studies as it is typical
β
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