1. ΔA(λ) = A(λ)LCPL - A(λ)RCPL
Circular Dichroism
ΔA(λ) = A(λ)LCPL - A(λ)RCPL

2. Polarized Light Light with oscillations confined to a single plane.
Top: vertically polarized light
Bottom: horizontally polarized light
Videos throughout notes from

3. Circularly Polarized Light
Sum of vertically and horizontally plane-polarized 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.

4. 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.

5. 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?

6. Origin of Ellipticity Unit
Would the signal above be positive or negative?

7. 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)
Backbone amide bond (far UV)
n-π* ~ nm (major peak in UV spectrum, (ε ~ 100 M-1cm-1)
π-π* ~190 nm
Sensitive to tertiary structure
Sensitive to secondary structure

8. UV Absorbance vs. CD: Poly-L-lysine
Random coil: positive 216nm
negative 198nm
Beta-sheet: negative 218nm
positive 195nm
Alpha-helix: negative 222nm
negative 208nm
positive 192nm
3 peaks from 2 electronic
transitions?
π-π*
n-π*
UV Absorbance: Cantor & Schimmel, 1980
CD: Campbell & Dwek, 1984
Both images from

9. Delocalized Excitation (Exciton)
Organic conformational analysis and stereochemistry from circular dichroism spectroscopy,
Lightner and Gurst, Wiley, 2000.

10. Spectroscopic Impact of Excitonic Coupling

11. Excitonic Coupling of Multiple Chromophores with Similar Excitation Energies (Examples)
head-to-tail dipole orientation
sandwich dipole orientation
800 nm
780 nm
820 nm
800 nm
790 nm
810 nm
From

12. Why is Excitonic Coupling Dependent on Secondary Structure?

13. 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?

14. 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:

15. Peptide Design Goal: ~60% helix
Tris-HCl (mM)
Ethanol (%)
TFE (%) °C
Helix (%)
Strand (%)
Turns (%)
Unordered (%)
Total (%) 5 7 9 77
100 50 22 15 14
101 10 36 12 18 34 20 44 16 30 3 99
How well did you estimate?

16. Other Uses of CD – Thermal Stability
Unfolded fraction (monitored at 222nm) = (θobs - θdenatured)/(θfolded - θdenatured)
Isoform
Melting Point (Tm), °C
Untreated
EDTA
Ca2+
Co2+
Ni2+
Zn2+
NPP2
58, 86
56, 102
56, 86
56,88 80 58
NPP6 76 70
N/A 68

17. Other Uses of CD – Absolute Stereochemistry of Chromophores
Organic conformational analysis and stereochemistry from circular dichroism spectroscopy,
Lightner and Gurst, Wiley, 2000.

18. 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-p-dimethylaminobenzoate derivative of trans-1,2-cyclohexanediol shows CD Δεmax,295 of -44 and Δεmax,320 of Which stereoisomer is this? (give both +/- chirality and R/S designation of both stereocenters)

19. Solution