1. Circular Dichroism Spectroscopy
Sourav Sarkar
2nd Year M.Sc student
Dept. of Chemistry
IIT Kharagpur, India.

2. What is CD Spectroscopy?
Circular Dichroism (CD) spectroscopy is a form of light absorption spectroscopy which is used in determining molecular behaviour in a broad spectrum of perturbation by measuring the difference in absorbance of right-circularly polarized (RPR) and left-circularly polarized (LCP) light.

3. Polarisation of Light Unpolarised light
(direction of oscillations randomly changes in a same plane with time)
Plane polarised light
(magnetic and electric field components oscillate in a definite plane being perpendicular to each other)
Circularly polarized light
(magnetic field remains oscillating but electric field vector changes direction in a rotary motion)

4. Circularly Polarised Light
Rotating electric field
Direction of propagation
Oscillating magnetic field

5. Source of Circularly Polarized Light1. 2.
Few Golden scrab beetle like Chrysina gloriossa and a few Mantis shrimp have been found to generate circularly polarized light.

6. Polarized Electromagnetic radiation Circularly polarized Linearly polarized Right hand(RCP) Left hand(LCP) [clockwise] [anticlockwise]

7. Mechanism of Action
1. Electric field of a light beam causes a linear displacement of charge when it interacts with a molecule. 2. Magnetic field of light beam causes circulation of charge. 3. These two motions combine to result an excitation of electrons in a helical fashion. 4. But the radiation used being chiral it interacts in different fashion with a definite chiral entity. 5. After interaction, the electric field vector traces out an elliptical path. So circularly polarized light becomes elliptical polarized light.

8. Photo elastic molulator
A Cartoon Diagram......
Short-arc Xe lamps
Rochon polarizer
Photo elastic molulator
Chiral specimen under expriment

9. What Actually Happens..??
1. When passing through an optical active medium, the speed between RCP and LCP light changes along with change in wavelength( ) and their absorptivity( ). 2. Now wavelength dependent difference of absorption(CD) is measured and plotted to get the CD spectra of the sample.

10. Molar circular dichroism
In few cases besides , Molar ellipticity is also measured.
Relation between and :
Where ER and EL are the magnitudes of the
electric field vectors of the RCP and LCP
light respectively.

11. Applications
To know whether the protein is folded or not. If so, then characterisation of its secondary, tertiary structure and its structural family.
Comparing the structure of a protein obtained from different sources.
To study the conformational stability of a protein under perturbation of environment caused by heat, pH, denaturants or stabilizer etc.
To study the effect of protein-protein or ligand-protein interaction.

12. CD Spectrum (200nm-250nm) (350nm-750nm) Far UV UV-Vis 100nm 950nm
Vacum UV Near UV Near IR
(100nm-200nm) (250nm-350nm) (750nm-950nm)

13. Classification..
Far UV-CD (200nm-250nm) is used to investigate the secondary structure of protein. Change in protein secondary structure as a function of temp.(T) or conc.(C) can be known. Which also allows us to know..
1. Enthalphy
2. Gibbs free energy of the process.
Near UV-CD (250nm-350nm) spectra provides information on tertiary structure. It provides information on the nature of the prosthetic group present in a protein.
UV-Vis CD (350nm-750nm) spectra in visible region is only found when metal is in chiral environment. Free metal ions in solution are not detected by Vis-CD. This can be used to detect protein bound metals.
Near IR-CD (750nm-950nm) can be used to investigate geometric and electronic structure by probing metal d-d transitions.

14. Case Study

15. Superimposed circular dichroism spectra of native and mutant S
Superimposed circular dichroism spectra of native and mutant S. coelicolor DHQase.
far UV
Superimposed circular dichroism spectra of native and mutant S. coelicolor DHQase. –, wild-type; - - -, R23A; · · ·, R23K; - · - ·, R23Q; A, far UV; B, near UV.
near UV
Krell T et al. J. Biol. Chem. 1996;271:
©1996 by American Society for Biochemistry and Molecular Biology

18. Ligand-DNA Interaction

19. Drug-Protein Interaction
Ibuprofen -HSA Diazepam-HSA
Foti, R. S., &Wahlstrom, J. L (12), pp. 900–905.

20. Interaction of Carmoisine With Serum Protein

21. C343-Anion With Serum Protein

22. Thermal Stability

23. Advantages Minimum quantity of sample is required.
Does not require extensive data processing.
Can be effectively used in presence of multiple co-factors.

24. Disadvantage & Complications
Carbohydrates can not be easily studied in CD.
Aqueous buffer system used in CD often absorb in the range where structural feature exist.
System should be completely devoid of O2 to perform the experiment below 200nm.
Buffer selectivity:
Phosphate, Sulphate, Carbonate, Acetate buffer can not be used until they are prepared in the range of 10-40mM.
Solvent selectivity:
A large number of organic solvents like THF, CHCl3, CH2Cl2 etc can not be used.
Lamp selectivity:
Instead of ordinary Xe-arc lamps, use of high pressure short arc Xe lamps are essential for doing low UV-CD spectroscopy.

25. Reference
Sharon M. Kelly, Thomas J. Jess, Nicholas C. Price; Biochimica et Biophysica Acta, 1751(2005),
Sharon M. Kelly, Nicholas C. Price; Biochimica et Biophysica Acta 1338(1997),
Datta S and Halder M; J. Phys. Chem. B, 118 (2014), 6071−6085.
Foti, R. S., & Wahlstrom, J. L.. 11(2008),
Krell T et al. J. Biol. Chem. 271(1996);

26. Thank you...