CD Spectroscopy

1
General features of spectroscopy
2
Dichroism
Linear Dichroism (LD)
Difference in absorption of versus ! polarized light
Optical Rotary Dispersion (ORD)
Rotation of linearly polarized light by sample
Circular Dichroism (CD)
Difference in absorption of left versus right circularly
polarized light
3
Polarization of Light
4
Polarization of Light
Plane-polarized light consists of two circularly polarized components
of equal intensity by opposite sense of rotation
the two components are like left- and right-handed helices
Two orthogonal plane waves with zero degree phase difference
form linearly polarized light, two orthogonal plane waves with a 90
phase difference form circularly polarized light. Depending on
whether the phase difference is plus or minus 90 the sense of
rotation is clockwise or anti-clockwise.
Differential absorption of the clock- and anti-clockwise components
by an optically active compound results in a rotation of the plane of
the light
5
Linearly and circular polarized light
6
Superposition of plane-polarized light
7
Differential absorption of two circularly
polarized beams
8
Cotton Effect
The differential absorption of circularly polarized light changes its sign in
the region of maximum absorption
ORD spectra are dispersive, CD spectra absoptive
9
Optical Rotatory Dispersion
the angular velocities of the left and right circularly polarized beams are
different in the sample. The orientation of the ellipse a is called optical
rotation. The measurement of the optical rotation in dependence of the used
wavelength is the optical rotatory dispersion.
(L) (R)
(L) (R)
10
Circular Dichroism
in substances with optical activity the left
and right circularly polarized light beams are
traveling at different speed and are
absorbed to a different extent.
the circular dichroism is characterized by
the ratio of the semiminor and semimajor
axes of the ellipse
tan= c/b
is known as the ellipticity
b
c
!
11
The components of a CD spectrometer
Block diagram of a spectropolarimeter (Jasco J-810). Plane polarized radiation is
produced by passage of light from the source (LS) through 2 prisms (P1 and P2) and a
series of mirrors (M0 to M5) and slits (S1 to S3). The ordinary ray (O) is focussed by a
lens (L), and passed through a filter (F) to the modulator (CDM). The circularly polarized
components are then passed through the shutter (SH) to the sample compartment,
before detection by the photomultiplier (PM).
12
Absorption and Molar Ellipticity
A() = A
l
() - A
r
() = [
l
() -
r
() ]!d!c = !d!c
c: concentration (mg/ml), d= path length (cm) (Lambert Beer)
A = /32.98
[] (molar ellipticity)= MW!100!/c!d (degcm
2
dmol
-1
) (c in mg/
ml; d in cm)
[] (mean residue ellipticity)= MRW!/10c!d (degcm
2
dmol
-1
)
MRW (mean residue weight): = M/(N-1) (approx. 1105 Da)
far UV: 100% helix content-> mean residue ellipticity at 222nm is
about -30000 (= -9 M
-1
cm
-1
)
near UV: mean residue ellipticity of aromatic side chains are less than
200 (less than 0.06 M
-1
cm
-1
)
13
Accurate knowledge of protein concentration
UV absorption at A
280
: (Trp or Tyr required)
A
280
= (5690
#(Trp)
+ 1280
#(Tyr)
+ 60
#(Cys)
)/MW
(For theoretical values see www.us.exapsy.org/tools/protparm.hmtl)
no contribution from light scattering
no other absorbing contaminant
correction applied for difference between native and folded
state (measure in ative buffer and in 8 M GdCl and compare the
A
280
values
14
Effects of Protein Concentration
CD spectra High-tension voltage traces
solid: 0.2 mg/ml
dotted: 1.0 mg/ml
dashed: 5.0 mg/ml
(lysozyme)
usable values < 700 V
15
Transitions of the amide bond

!
o
->!* : 190 nm
electrically allowed,

max
of 10
4
M
-1
cm
-1
transition moment along
N-O direction
solvent insensitive

n-> !* : 215-222 nm
electrically forbidden,

max
of 100 M
-1
cm
-1
large transition dipole
moment along the
carbonyl bond

solvent sensitive
The intensity and energy of these transitions
depends on these transitions depends on and
(secondary structure)
16
CD and Secondary Structure
\vz}z_(j, q
190 200 210 220 230 240 250
[
!
]

0
-
3
,

,
z
_
,
,
q

,
,
q
z
}
-

-60
-40
-20
0
20
40
60
80
"#jz};
$-sheet
|ypz ((;
Random coil
Poly-L-proline (P2)
Accuracy of the CD method
(compared to known structures):
helix 95-100%
sheet < 75%
turn < 25%
other < 90 %
17
Typical CD spectra of regular secondary structures
-helix -sheet
-turn
rc
PP-2 helix
An all helix polypeptide
has an ellipticity of
-38000 deg cm
2
mol
-1
res
-1
at 222 nm
An all random polypeptide
has an ellipticity of -1200
deg cm
2
mol
-1
res
-1
at 222
nm
In general, the CD signal
at 215 nm indicates the
sheet content and the
signal at 208 nm and 222
nm are used to calculate
the helical content.
18
Features of CD spectra
Secondary
structure element
Signal Electron
transition
Position of minimum or
maximum
Molar ellipticity of minima
and maxima
[degcm
2
dmol
-1
]
-helix positive -> * 190-195 nm 60.000 to 80.000
negative -> * 208 -36.000 3.000
negative n-> * 222 -36.000 3.000
-sheet positive -> * 195 - 200 30.000 to 50.000
negative n-> * 215 - 220 -10.000 to 20.000
random negative -> * ca. 200 -20.000
positive n-> * 220
19
Estimation of Secondary Structure (Content)
In a first approximation, a CD spectrum of a protein or polypeptide
can be treated as a sum of three components: -helical, -sheet,
and random coil contributions to the spectrum.
At each wavelength, the ellipticity () of the spectrum will contain a
linear combination of these components:

tot
=
h

h
+
S

S
+
C

C

tot
is the total measured elipticity,
h
the contribution from helix,

s
for sheet,
c
for coil, and the corresponding the fraction of this
contribution.
The experimental spectrum can be back-calculated from the
individual contributions, and the deviation across all ellipticities be
minimized.
20
Contributions of !-Systems to CD Spectra
The near UV CD spectrum for type II dehydroquinase from Streptomyces coelicolor. The
wavelength ranges corresponding to signals from Phe, Tyr and Trp side chains are indicated,
but it should be emphasized that there can be considerable overlap between the Tyr and Trp
signals.
Far-UV CD spectra are complicated by Phe, Tyr, Trp and S-S bonds that
can dominate that region because of allowed !->!* transitions
21
Contributions of !-Systems to CD Spectra (II)
CD spectra of wild type and mutant (R23Q) type II
dehydroquinase from Streptomyces coelicolor.
The far UV spectrum (panel A) and near UV spectrum
(panel B) show that the wild-type (solid line) and mutant
(dotted line) enzymes have very similar secondary and
tertiary structures. The loss of catalytic activity in the
mutant cannot therefore be due to an inability to acquire
the correct folded structure.
22
The choice of the Buffer
Absorption properties of selected bufer components in the far UV
Component Absorbance (50 mM solution in 0.02 cm pathlength cell)
180 nm 190 nm 200 nm 210 nm
NaCl >0.5 >0.5 0.02 0
NaF 0 0 0 0
NaClO
4
0 0 0 0
Boric acid 0 0 0 0
Na borate
(pH 9.1)
0.3 0.09 0 0
Na
2
HPO
4
>0.5 0.3 0.05 0
NaH
2
PO
4
0.15 0.01 0 0
Na acetate >0.5 >0.5 0.17 0.03
Tris/H
2
SO
4
(pH 8.0)
>0.5 0.24 0.13 0.02
HEPES/Na
+
(pH 7.5)
>0.5 >0.5 0.5 0.37
MES/Na
+
(pH 6.0)
>0.5 0.29 0.29 0.07
23
The choice of the Buffer (II)
The effects of buffer components on far UV CD spectra. Lysozyme (0.2 mg/ml) was dissolved in 50 mM sodium
phosphate buffer, pH 7.5 (spectrum 1, solid line), or sodium phosphate buffer containing either 150 mM NaCl
(spectrum 2, dashed line) or 150 mM imidazole (spectrum 3, dash dot dot line), or in 50 mM Tris/acetate, pH 7.5
(spectrum 4, dotted line).
24
Maintenance
(quartz) cells are expensive (400$), handle with care, dont scratch
with pipette.
quartz cell should be washed. Use conc. HNO
3
for 1 min, in bad case
for 1 h, followed by extensive washing with distilled water, then
ethanol, followed by drying with vacuum pump (or blow N
2
gas
through them). Dont use pressurized air since it contains traces of
oil.
purge instrument with N
2
before usage (O
2
will be converted by the
UV to the aggressive O
3
.
after switching on allow for 30 min to warm up. Check stability of
the instrument by the drift of the baseline.
lifetime of the light source is approx. 1000 h of usage, after which
output will be poorer.
25
CD and conformational changes
Monitoring
222
of a protein as a function of temperature or
chemical denaturant yields important information on protein
stability allowing to compute the thermodynamic parameter G
u
,
H
u
, S
u
, T
m
and C
p
-16
-14
-12
-10
-8
-6
-4
0 10 20 30 40 50 60 70 80 90 100
T [C]
-8000.0
-7000.0
-6000.0
-5000.0
-4000.0
-3000.0
-2000.0
-1000.0
0.0
1000.0
200 210 220 230 240 250
nm
m
d
e
g
Secondary structure
prediction (K2D):
- -helix: 26%
- -sheet:21%
Unfolding of Procaspase-8
26
Montoring pH-induced Denaturation
pH-Induced denaturation of natively folded HuIL-1!
27
TFE-induced folding of natively unfolded -
-synuclein
TFE conc.
28
Cation-binding induced folding
Uversky et al. (2000) BBRC 267, 663
Uversky et al. (2001) JBC 276, 44284
Uversky et al. (2002) JPR 1, 149
Permyakov et al. (2003) Proteins 53, 855
Munishkina et al. (2004) JBC 342, 1305
Zn
2+
conc.
29
220 240 260 280 300 320
-400
-200
0
200
[
!
]

x

1
0
-
3

[
d
e
g

c
m

2

d
m
o
l

-
1
]
wavelength [nm]
-2 0 2 4 6 8 10
0,0
0,2
0,4
0,6
0,8
1,0
l
n

(
a
b
s
o
r
p
t
i
o
n
2
4
0
n
m
)

[
O
D
]
Cd(II) / Thionein
a
b
s
o
r
p
t
i
o
n
2
4
0
n

(
n
o
r
m
a
l
i
z
e
d
)
220 240 260 280 300 320
-400
-200
0
200
wavelength [nm]
0 2 14 16 18
-3,0
-2,5
-2,0
-1,5
-1,0
Time [h]
Cd
2+
Cd
2+
time course of the
metal-loading reaction
followed at 240 nm.
Metal titration of blue-crab MT followed by CD
30