CD Spectroscopy
1 General features of spectroscopy
–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
• in substances with optical activity the left b θ and right circularly polarized light beams are traveling at different speed and are absorbed to a different extent. • the circular dichroism is characterized by c 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, 4 -1 -1 εmax of 10 M cm transition moment along N-O direction solvent insensitive
•n-> π* : 215-222 nm electrically forbidden, -1 -1 εmax of 100 M cm large transition dipole The intensity and energy of these transitions moment along the depends on these transitions depends on φ and carbonyl bond solvent sensitive ψ(secondary structure)
16 CD and Secondary Structure
80 1 - l 60 a-helix
o b-sheet Type 1 turn m
. 40 2 Poly-L-proline (P2) Accuracy of the CD method m
c (compared to known structures):
. 20
g helix 95-100% e d
0 sheet < 75% , 3
- turn < 25% 0 -20 1 other < 90 %
X
] -40 q [
-60 190 200 210 220 230 240 250 Wavelength, nm
17 Typical CD spectra of regular secondary structures
An all helix polypeptide has an ellipticity of -38000 deg cm2mol-1res-1 at 222 nm α-helix β-sheet
An all random polypeptide β-turn has an ellipticity of -1200 PP-2 helix deg cm2mol-1res-1 at 222 nm rc 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 Electron Position of minimum or Molar ellipticity of minima structure element transition maximum and maxima [deg·cm2 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: 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 bu!er 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 0.3 0.09 0 0 (pH 9.1) Na2HPO 4 >0.5 0.3 0.05 0 NaH2PO4 0.15 0.01 0 0 Na acetate >0.5 >0.5 0.17 0.03 Tris/H 2SO4 >0.5 0.24 0.13 0.02 (pH 8.0) HEPES/Na + >0.5 >0.5 0.5 0.37 (pH 7.5) MES/Na + >0.5 0.29 0.29 0.07 (pH 6.0)
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
1000.0 -4
0.0 200 210 220 230 240 250 -6 -1000.0
-8 -2000.0
-3000.0 -10 mdeg -4000.0 -12 -5000.0 Secondary structure -6000.0 prediction (K2D): -14 -7000.0 - α-helix: 26% -16 -8000.0 - β-sheet:21% 0 10 20 30 40 50 60 70 80 90 100 nm 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 Metal titration of blue-crab MT followed by CD ] -1
200 2+ 200 2+ dmol
2 Cd Cd
0 0 [deg cm -3 0 -200 -200 ] x 1 θ [ -400 -400
220 240 260 280 300 320 220 240 260 280 300 320 wavelength [nm] wavelength [nm]
-1,0 1,0
-1,5 0,8 ) [OD] 240nm 0,6 n (normalized) -2,0 time course of the 240n n 0,4 metal-loading reaction -2,5 followed at 240 nm. 0,2 ln (absorptio absorptio 0,0 -3,0
-2 0246810 0214 16 18 Cd(II) / Thionein Time [h]
30