CD spectroscopy
陳佩燁
Rita P.Y. Chen
中央研究院生化所
Circular dichroism spectroscopy
1
2
Far UV CD
n -> π* (210-230 nm) centered around 220 nm,
ε = ~100 mol-1 dm3 cm-1,
involves non-bonding electrons of O of the carbonyl
π -> π* centered around 190 nm
ε = ~7000 mol-1 dm3 cm-1,
π -> π*dominated by the carbonyl π-bond, and also affected
by the involvement of the nitrogen in the π-orbitals
The intensity and energy of these transitions depends on φ
and ψ (i.e., secondary structure)
exiton coupling of the π->π* transitions leads to
positive (π−>π*)perpendicular (to helix axis) at
192 nm and negative (π−>π*)parallel at 209 nm
negative at 222 nm is red shifted (n->π*)
3
-1
-3
2
[θ] X 10 , de g . cm . dmol
80
Negative at 222 nm (n -> π*)and 208 nm (π−>π*)
Positive at 192 nm (π−>π∗)
α−helix
β-sheet
Type 1 turn
Random coil
Poly-L-proline (P2)
60
40
20
0
-20
-40
-60
190
200
210
220
230
240
250
Wavelength, nm
Poly(Pro)I, cis peptide bonds, right-handed helix, 3.3 residue/turn, in propanol
Poly(Pro)II, trans peptide bonds, left-handed helix, 3.0 residue/turn, in water
Beer-Lambert law
A= log10 (Iin/Iout)
A= ε x b x c
∆ε = εL-εR differential absorbance of a 1 mol/l solution in a 1 cm cell
Measured θ , ellipticity, is the rotation in degrees
of a 1dmol/cm3 solution and a pathlength of 1 cm
Molar ellipticity: [θ] =
degrees cm2 dmol-1
θ x 100 x Mr
cx l
c: mg/ml
l: cm
Mean residue ellipiticity: [θ] MRW = [θ] /residue number
degrees cm2 dmol-1 residue -1
∆ε = [θ] /3298
Litre mol-1 cm-1 or M-1 cm-1
4
Use far-UV CD to determine amounts of secondary
structure in proteins
generate basis sets by determining spectra of pure α-helix, β-sheet, etc. of synthetic
peptides
or deconvoluting CD spectra of proteins with know structures to generate basis sets of
each of secondary structure
poly-L-lysine {(Lys)n} can adopt 3 different conformations merely by varying the pH and
temperature
random coil at pH 7.0
α-helix at pH 10.8
β-form at pH 11.1 after heating to 52°C and recooling
CD spectrum of unknown protein = fαSa(l) + fβSb(l) + fRCSRC(l), where Sa(l), Sb(l), and
SRC(l) are derived from poly-L-lysine basis spectra.
5
The disadvantage of this method is that although these basis sets are easily
determined by direct measurement, they do not always agree from one lab to
another. In addition, chain length and aggregation effect the basis set spectra.
However, this method is usually accurate to within 10% for α-helix content.
Technique
X-ray
CD using
(Lys)n Basis
Sets
Secondary
Structure
carboxypeptid
ase
αchymotrypsin
myoglobin
α
23%
8%
68%
β
18%
22%
0%
RC + other
59%
70%
32%
α
13%
12%
68%
β
31%
23%
5%
RC + other
56%
65%
27%
Error of deconvolution might come
from…..
6
Protein structure IRL press
Far-UV CD
Near-UV CD
7
Near UV CD
Wavelength Range
250-270nm
270-290nm
280-300nm
250-350nm
Chromophore Contributions
side chain Phe
side chain Tyr
side chain Trp
disulphide bond
Two possible transition dipole moments for Trp
Protein thermal stability
8
Protein chemical stability
1400
intensity at 303 nm
yF=1189+80.6x[GdnHCl]
1200
1000
800
600
U
Kfold
yU=319+40.3x[GdnHCl]
F
400
0
1
2
3
4
GdnHCl (M)
5
6
7
9
10