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

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.

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)

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

Source of Circularly Polarized Light 1. 2. Few Golden scrab beetle like Chrysina gloriossa and a few Mantis shrimp have been found to generate circularly polarized light.

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

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.

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

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.

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.

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.

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

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.

Case Study

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;271:24492-24497 ©1996 by American Society for Biochemistry and Molecular Biology

Ligand-DNA Interaction

Drug-Protein Interaction Ibuprofen -HSA Diazepam-HSA Foti, R. S., &Wahlstrom, J. L. 2008. 11(12), pp. 900–905.

Interaction of Carmoisine With Serum Protein

C343-Anion With Serum Protein

Thermal Stability

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

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.

Reference Sharon M. Kelly, Thomas J. Jess, Nicholas C. Price; Biochimica et Biophysica Acta, 1751(2005), 119-139. Sharon M. Kelly, Nicholas C. Price; Biochimica et Biophysica Acta 1338(1997), 161-185. Datta S and Halder M; J. Phys. Chem. B, 118 (2014), 6071−6085. Foti, R. S., & Wahlstrom, J. L.. 11(2008), 900-905. Krell T et al. J. Biol. Chem. 271(1996); 24492-24497.

Thank you...