Circular Dichroism in Protein Analysis
69
of the two long-wavelength bands, their
ratio, and the wavelength of the positive
band. These can be affected by side chains,
solvents, and other environmental factors.
Structural factors may also be important;
β
-sheets can be antiparallel, parallel, or
mixed, intra- or intermolecular, and are
also twisted to varying extents. Theoretical
studies suggest that the extent of twisting
of
β
-sheets is probably more important
than the distinction between antiparallel
and parallel sheets.
The wavelength at which the intensity
of the CD spectrum changes sign is the
most convenient criterion for differenti-
ating parallel and antiparallel
β
-sheets.
Crossover at
178 nm indicates antipar-
allel
β
-sheet, at
192 nm, parallel
β
-sheet,
and at
187 nm, a mixture of parallel and
antiparallel
β
-sheets. The effects of twist-
ing on
β
-sheet CD are predicted to include
large differences in the magnitudes of the
two long-wavelength bands, and a red-shift
of both the positive maximum and the
short-wavelength crossover from positive
to negative CD.
Unlike
α
-helix content, it is not possible
to estimate
β
-sheet content in proteins
reliably
from
CD
spectra
because
of
the twisted and planar
β
-sheet showing
quantitative differences between parallel
and antiparallel
β
-sheet. One method is to
calculate the difference between the CD at
217 nm and 195 nm. For 100%
β
-sheet, it
is50–55
×
10
3
deg cm
2
dmol
1
.Thistype
of estimate may be reasonable, as long as
the
β
-sheet is not strongly twisted.
6.4
β
-turn
There are eight types of
β
-turn, three of
wh
icha
recommoninp
ro
te
ins
.TheCD
spectra of
β
-turn are rather varied. The
type II
β
-turn has a strong negative band
between 180 and 190 nm, a strong positive
band between 200 and 205 nm, and a
weak, red-shifted band at 225 nm due to
the n
π
transition. Generally, type II CD
spectra are similar to
β
-sheet CD, except
that the maximum is red-shifted by 5 to
10 nm. The CD spectra for type I and III
turns are similar to that of the
α
-helix,
with a negative n
π
band and a negative
ππ
couplet.
6.5
Random Coil
CD spectroscopy is sensitive to precise
protein conformation, but most random
coils are very flexible. So, there are less
common features in their CD spectra, ex-
cept a strong negative band near 200 nm
and a weak band at longer wavelengths,
but the latter band may be either posi-
tive or negative. Poly(Glu) and poly(Lys)
are frequently used models of unordered
polypeptides. Their neutral aqueous solu-
tion spectra show two characteristic fea-
tures: a strong negative band near 200 nm
([
θ
]
max
∼−
40 000 deg cm
2
dmol
1
)anda
signi±cant band shift between 200 nm and
191 nm. When the pH is set to neutralize
the side chains, or the ionic strength is in-
creased by added salt, or the temperature
is increased, the very weak negative band
in the 235- to 240-nm region is observable.
Urea and guanidinium chloride (GuCl)
can have a complicated effect on CD spec-
tra. At low concentrations, GuCl causes a
decrease in the long-wavelength CD bands
of the charged polypeptides by shielding
th
ech
a
rg
e
sonth
es
id
ech
a
in
sandd
e
-
creasing the electrostatic stabilization of
the helical peptide.
There are two different explanations for
unordered polypeptides CD spectra. In the
conventional view, random coil polypep-
tides exist as an ensemble of an enormous
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