Circular Dichroism in Protein Analysis
61
transitions on a single peptide group may
mix – an interaction known as the
one-
electron effect
.Inaninfn
i
tehe
l
ix
,exc
i
ton
interactions oF
ππ
transitions on diFFer-
ent peptide groups lead to three allowed
transitions, one polarized parallel to the
helix axis and two degenerate (equal en-
ergy) bands polarized perpendicular to the
helix axis.
2.2
Near Ultraviolet
The near UV region, 250 to 300 nm, is
also called the
aromatic region
.Becausethe
peptide absorption in this area is at least
an order oF magnitude weaker than in the
Far UV region, a small number oF aromatic
chromophores have a distinct advantage.
B
a
n
d
si
nt
h
i
sr
e
g
i
o
na
r
ed
u
et
o
ππ
transitions From the aromatic side chains
oF histidine, phenylalanine, tyrosine, and
tryptophan and n
σ
transitions From disul-
fdegroups
.Theindo
leoFtryp
tophanhas
two or more transitions in the range oF 240
to 290 nm with a combined maximum
extinction
coeFfcient
ε
max
(
279 nm
)
5600 dm
3
cm
1
mol
1
;t
y
r
o
s
i
n
eh
a
s
one
transition
with
ε
max
(
274 nm
)
1400 dm
3
cm
1
mol
1
and phenylalanine
has one transition with
ε
max
(
258 nm
)
190 dm
3
cm
1
mol
1
. The cystine disul-
fde bond absorbs From 250 to 270 nm with
ε
max
300 dm
3
cm
1
mol
1
.T
h
en
e
a
r
UV CD also reflects the environment oF
aromatic groups. ±or example, the most
red-shiFted tyrosine Feature should be due
to tyrosine(s) in a highly polarizable envi-
ronment, while the most blue-shiFted are
duetoexposuretosolvent
.A
lthoughtryp-
tophan has the most intense transition in
this region, usually proteins have Few tryp-
tophans compared to the other aromatic
groups, so the region tends not to be dom-
inated by tryptophan transitions.
The
disulfde
group
oF
cystine
in
many extracellular proteins has two well-
characterized n
σ
electronic transitions in
the near UV region. The wavelengths de-
pend on the dihedral angle oF the disulfde
band. This dihedral angle is normally ap-
proximately 90
in proteins, and the two
n
σ
transitions are degenerate, giving a
single broad absorption band near 260 nm.
As the dihedral angle deviates From 90
,
the degeneracy is broken and one tran-
sition shiFts to higher energy, with the
other moving to lower energy. Disulfde
contributions to a near UV CD spectrum
are distinguishable From those oF aro-
matic side chains, because the Former are
much broader.
3
Vibrational Circular Dichroism
Vibrational
optical
activity
consists
oF
two complementary spectroscopic areas,
vibrational circular dichroism, and vi-
brational Raman optical activity. Both
spectra show the diFFerential response to
leFt- versus right-circularly polarized ra-
diation due to a vibrational transition
in a chiral molecule. Vibrational circular
dichroism (VCD) arises From simultane-
ous changes in the electric and mag-
netic dipole moments oF the molecule
due to the nuclear motion. The vibra-
tional transition is between the g
0
and
g
1
vibrational sublevels oF the ground
electronic state. The transition dipole cou-
pling is highly dependent on peptide
secondary structure. Because the interpre-
tation oF VCD depends on band shape, it is
less susceptible to error From Frequency
shiFts
arising
From
nonstereochemical
sources.
The amide I band (amide I
0
in D
2
O),
due to primary C
O stretch, is the most
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