Circular Dichroism
ΔA(λ) = A(λ)LCPL - A(λ)RCPL
Polarized Light
• Light with
oscillations
confined to a single
plane.
• Top: vertically
polarized light
• Bottom:
horizontally
polarized light
Videos throughout notes from http://www.photophysics.com/polarization.php
Circularly Polarized Light
• Sum of vertically and horizontally planepolarized light in which the phases differ by a
quarter wave
•Does this video represent left or right-handed circularly polarized light?
•Draw separate 2D representations for the vertical and horizontal components that
clearly show the phase difference.
•Diagram the phase different required to obtain the opposite direction of circularly
polarized light.
Circular Dichroism (CD) Spectroscopy
• Difference in absorption between left-handed
and right-handed circularly polarized light as a
function of wavelength
• Unlike optical rotatory dispersion (ORD)
spectroscopy, which only requires a chiral
molecule, CD requires a chromophore in a
chiral environment.
ORD – why isn’t a chromophore required?
• All chiral molecules exhibit circular birefringence, basically the
rates of propagation of left and right-handed polarized light
(which can be added to form plane-polarized light) are
different. This changes their relative phase, and therefore the
observed angle at the detector.
With the background given so far, how would the above video change to represent CD?
Origin of Ellipticity Unit
Would the signal above be positive or negative?
Chromophores in Biomolecules
• Proteins
– Aromatic sidechains (π-π* ~280 nm, near UV)
• Phenylalanine (ε ~ 250 M-1cm-1)
• Tyrosine (ε ~ 1000 M-1cm-1)
• Tryptophan (ε ~ 5000 M-1cm-1)
Sensitive to tertiary
structure
– Backbone amide bond (far UV)
• n-π* ~210-220 nm (major peak in UV spectrum, (ε ~
100 M-1cm-1)
Sensitive to
secondary structure
• π-π* ~190 nm
UV Absorbance vs. CD: Poly-L-lysine
Random coil: positive CD @ 216nm
negative CD @ 198nm
Beta-sheet: negative CD @ 218nm
positive CD @ 195nm
Alpha-helix: negative CD @ 222nm
negative CD @ 208nm
positive CD @ 192nm
π-π*
n-π*
3 peaks from 2 electronic
transitions?
UV Absorbance: Cantor & Schimmel, 1980
CD: Campbell & Dwek, 1984
Both images from http://www.cryst.bbk.ac.uk/PPS2/course/section8/ss-960531_21.html
Delocalized Excitation (Exciton)
Organic conformational analysis and stereochemistry from circular dichroism spectroscopy,
Lightner and Gurst, Wiley, 2000.
Spectroscopic Impact of Excitonic
Coupling
Excitonic Coupling of Multiple
Chromophores with Similar Excitation
Energies (Examples)
head-to-tail dipole orientation
sandwich dipole orientation
780 nm
790 nm
800 nm
800 nm
820 nm
810 nm
From http://www.chem.vu.nl/~zwan/lectures/Egmond_3.ppt
Why is Excitonic Coupling Dependent
on Secondary Structure?
CD: Reflection of Environment on
Protein Folding
Which traces suggest random coil structure?
What secondary structure type is suggested by the other traces?
Can you estimate the fraction random coil or other secondary structure?
Near-UV CD Data Analysis
• Deconvolution: determining contributions to
observed spectrum from separate secondary
structures
• Algorithms (most available through Dichroweb)
– Singular value decomposition: determines
coefficients for a matrix of basis spectra of known
secondary structure
– Self-consistent (SELCON3): initial guess by SVD
followed by iteration to self-consistent result
– K2D: neural network trained using CD/structural data
on reference proteins to translate input CD spectra to
secondary structure proportions
Dichroweb: http://dichroweb.cryst.bbk.ac.uk/
Peptide Design Goal: ~60% helix
Tris-HCl
(mM)
Ethanol
(%)
TFE
(%)
°C
Helix
(%)
Strand
(%)
Turns
(%)
Unordered
(%)
Total (%)
0
0
0
5
7
9
7
77
100
50
0
0
5
22
15
14
50
101
50
5
0
10
36
12
18
34
100
50
20
0
5
44
10
16
30
100
0
0
20
5
77
3
5
14
99
How well did you estimate?
1.2
1.2
1.0
1.0
NPP6 Unfolded Fraction
NPP2 Unfolded Fraction
Other Uses of CD – Thermal Stability
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
0.0
0.0
-0.2
-0.2
20
30
40
50
60
70
80
90
100
20
110
30
40
50
60
70
80
90
Temperature (°C)
Temperature (oC)
Unfolded fraction (monitored at 222nm) = (θobs - θdenatured)/(θfolded - θdenatured)
Melting Point (Tm), °C
Isoform
Untreated
EDTA
Ca2+
Co2+
Ni2+
Zn2+
NPP2
58, 86
56, 102
56, 86
56,88
80
58
NPP6
76
76
70
N/A
N/A
68
100
110
Other Uses of CD – Absolute
Stereochemistry of Chromophores
Organic conformational analysis and stereochemistry from circular dichroism spectroscopy,
Lightner and Gurst, Wiley, 2000.
Data Interpretation Problem
• Given: p-Dimethylaminobenzoate has an
electronic transition dipole running through
the substituents with a UV εmax,310 of ~30,000.
• One stereoisomer of the bis-pdimethylaminobenzoate derivative of trans1,2-cyclohexanediol shows CD Δεmax,295 of -44
and Δεmax,320 of +83. Which stereoisomer is
this? (give both +/- chirality and R/S
designation of both stereocenters)
Solution