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
(L)
(R)
(L)
(R)
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.
10
Circular Dichroism
bθ
c
•
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
tanθ= c/b
•
θ is known as the ellipticity
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
ΔA(λ) = Al(λ) - Ar(λ) = [εl(λ) - εr(λ) ]✕d✕c = Δε✕d✕c
c: concentration (mg/ml), d= path length (cm) (Lambert Beer)
ΔA = θ/32.98
[θ] (molar ellipticity)= MW✕100✕θ/c✕d (deg•cm2•dmol-1) (c in mg/
ml; d in cm)
[θ] (mean residue ellipticity)= MRW✕θ/10c✕d (deg•cm2•dmol-1)
MRW (mean residue weight): = M/(N-1) (approx. 110±5 Da)
far UV: 100% helix content-> mean residue ellipticity at 222nm is
about -30000 (Δε= -9 M-1 cm-1)
near UV: mean residue ellipticity of aromatic side chains are less than
200 (Δεless than 0.06 M-1 cm-1)
13
Accurate knowledge of protein concentration
•
UV absorption at A280: (Trp or Tyr required)
– A280= (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
A280 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
•πo->π* : 190 nm
electrically allowed,
εmax of 104M-1cm-1
transition moment along
N-O direction
solvent insensitive
•n-> π* : 215-222 nm
electrically forbidden,
εmax of 100 M-1cm-1
The intensity and energy of these transitions
depends on these transitions depends on φ and
ψ(secondary structure)
large transition dipole
moment along the
carbonyl bond
solvent sensitive
16
CD and Secondary Structure
-3 , de g . cm 2 . dmol-1
X
10
[q]
80
a-helix
b-sheet
Type 1 turn
Random coil
Poly-L-proline (P2)
60
40
Accuracy of the CD method
(compared to known structures):
helix 95-100%
sheet < 75%
turn < 25%
other < 90 %
20
0
-20
-40
-60
190
200
210
220
230
240
250
Wavelength, nm
17
Typical CD spectra of regular secondary structures
α-helix
β-sheet
β-turn
PP-2 helix
rc
An all helix polypeptide
has an ellipticity of
-38000 deg cm2mol-1res-1
at 222 nm
An all random polypeptide
has an ellipticity of -1200
deg cm2mol-1res-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
Signal
structure element
Electron
Position of minimum or
Molar ellipticity of minima
transition
maximum
and maxima
[deg·cm2 dmol-1]
-helix
-sheet
random
positive
-> *
190-195 nm
60.000 to 80.000
negative
-> *
208
-36.000 ± 3.000
negative
n-> *
222
-36.000 ± 3.000
positive
-> *
195 - 200
30.000 to 50.000
negative
n-> *
215 - 220
-10.000 to –20.000
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: α-helical, β-sheet,
and random coil contributions to the spectrum.
• At each wavelength, the ellipticity (θ) of the spectrum will contain a
linear combination of these components:
θtot = χh• θh + χS• θS + χC• θC
• θtot is the total measured elipticity, θh the contribution from helix,
θs for sheet, θc for coil, and the corresponding χthe 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 π-Systems to CD Spectra
Far-UV CD spectra are complicated by Phe, Tyr, Trp and S-S bonds that
can dominate that region because of allowed π->π* transitions
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.
21
Contributions of π-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 buffer components in the far UV
Component
NaCl
NaF
NaClO 4
Boric acid
Na borate
(pH 9.1)
Na2HPO 4
NaH 2PO 4
Na acetate
Tris/H 2SO 4
(pH 8.0)
HEPES/Na +
(pH 7.5)
MES/Na +
(pH 6.0)
Absorbance (50 mM solution in 0.02 cm pathlength cell)
180 nm
190 nm
>0.5
0
0
0
0.3
>0.5
0
0
0
0.09
0.02
0
0
0
0
0
0
0
0
0
0.3
0.01
>0.5
0.24
0.05
0
0.17
0.13
0
0
0.03
0.02
0.5
0.37
0.29
0.07
>0.5
0.15
>0.5
>0.5
>0.5
>0.5
>0.5
0.29
200 nm
210 nm
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, don’t scratch
with pipette.
•
quartz cell should be washed. Use conc. HNO3 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 N2 gas
through them). Don’t use pressurized air since it contains traces of
oil.
•
purge instrument with N2 before usage (O2 will be converted by the
UV to the aggressive O3.
•
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 θ222 of a protein as a function of temperature or
chemical denaturant yields important information on protein
stability allowing to compute the thermodynamic parameter ΔGu,
ΔHu, ΔSu, Tm and ΔCp
Unfolding of Procaspase-8
-4
1000.0
0.0
200
-1000.0
210
220
230
240
250
-8
-2000.0
mdeg
-6
-3000.0
-10
-4000.0
Secondary structure
prediction (K2D):
- α-helix: 26%
-5000.0
-6000.0
-7000.0
-8000.0
nm
- β-sheet:21%
-12
-14
-16
0
10
20
30
40
50
60
70
80
90
100
T [ºC]
26
Montoring pH-induced Denaturation
pH-Induced denaturation of natively folded HuIL-1β
27
TFE-induced folding of natively unfolded α-synuclein
TFE conc.
28
Cation-binding induced folding
Zn2+ conc.
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
29
-1
[θ] x 10 [deg cm dmol ]
Metal titration of blue-crab MT followed by CD
Cd2+
2
200
Cd2+
200
0
-200
-200
-400
-400
-3
0
220
240
260
280
300
320
220
wavelength [nm]
240
260
280
300
320
wavelength [nm]
ln (absorption240nm) [OD]
absorption240n (normalized)
-1,0
1,0
-1,5
0,8
0,6
time course of the
metal-loading reaction
followed at 240 nm.
-2,0
0,4
-2,5
0,2
-3,0
0,0
-2
0
2
4
6
Cd(II) / Thionein
8
10
0
2
14
16
18
Time [h]
30