CD Spectroscopy Presentation1.pptx

How to determine the structure of proteins by using Circular Dichorism.

1. CIRCULAR DICHROISM Iqra Shahzadi PhD Student

2. Why use CD? Information on molecular and electronic structure. Experiments are relatively quick and easy to perform. Non- destructive (can recover most samples). A d v a n t a g es Simple and quick e xpe rim e n ts No extensive preparation Meas u r e m e n t on solution phase Any size of mac r omole c u le Mi c r o s e c ond time resolution Relatively low concentration /amounts of sample Figure: Advantages of CD Spectropolarimeter 2 4. Solution phase. Crystallization process could change molecular structure. 5. Low concentrations (0.1 mg/mL). Doesn’t require a concentration which could change the system being studied.

3. Circular dichroism is the difference in the absorption of left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL). Occurs when a molecule contains one or more chiral chromophores. Intrinsically chiral Covalently linked to chiral center Placed in asymmetric environment Circular dichroism = ΔA(λ) = A(λ) LCPL - A(λ) RCPL (where λ is the wavelength) What is Circular Dichroism? 3 Cordierite

4. Chiral molecules https://socratic.org/organic-chemistry-1/r-and-s-configurations/chiral-and-achiral-molecules- 1 N C C H H R O O H Side Chain Carb o xyl Group H Amino Group Figure: General Structure of Amino Acid Chiral Z wi t t er Ion Polarity d e p e nd e n t on side chain Change in protein structure alters the polarity 4

5. Electromagnetic wave http://www.emagtech.com/wiki/index.php?title=File:EM_wave.png Light path Amplitude Amplitude : intensity of wave from tip of crest to central axis Wavelength : distance between two consecutive crests Polarization : directionality of electric and magnetic fields 5 x z y Unpolarised A Polarization of Light y x z Linearly or Plane Polarised B Left Handed Right Handed C i r c ular l y Polarised C Figure: Schemes of the electric field components of unpolarized .

6. Left Circularly Polarized light Right Circularly Polarized light 6 Vertically polari z ed light Horizontally polarized light

7. ∆𝐴 = 𝐴 𝐿 − 𝐴 𝑅 LH RH  Elliptically polarized light Elliptically polarized light : electric field components rotate along beam propagation, perpendicular, unequal in amplitude , 90° phase difference Circularly polarized light : electric field components rotate along beam propagation, perpendicular, equal in amplitude , 90° phase difference 7

8. Circular Dichroism Some materials possess a special P roperty . Assume that a plane-polarized light wave (blue) traverses a medium that does not absorb the left circularly polarized component (red) of the wave at all but highly absorbs the right circularly polarized component (green). The intensity of the green component decreases I n comparison to the red one. The superposition of the two components yields a resulting field vector that rotates along an ellipsoid path and is called an elliptically polarized light. 8 Optically active molecules absorb left handed and right handed light differentially. This difference in absorbance is called CIRCULAR DICHROISM or CD. Optically active mole c ules Sa m ple Left Handed Right Handed C i r c ular l y Polarised Figure: Schematic Representation of Circular Dichroism

9. JASCO Corporation Product JASCO Corporation Hachioji, Tokyo Since 1958 9

10. Principle of Measurement Light Source Unpolarized light Linearly polarized light PEM Circularly polarized light Double Monochromator Sample PMT Detector RCP LCP RCP LCP Prisms 10

11. CD Spectrum 100 nm 950 nm Near IR 750-950 nm UV-Vis 350-750 nm Near UV 250-350 nm Vacuum UV 100-200 nm Far UV 200-250 nm Am ide, Secondary Structure Aromatic groups, Tertiary structure, Folding Extrinsic groups, Tertiary structure, Ligand binding Figure: Schematic Representation of Classification of CD Spectrum Regions 11

12. CD Spectra of Protein Secondary Structures -ve band (nm) +ve band (nm) α-helix 222 192 208 β-sheet 216 195 Random coil 200 Figure : Fasman Standard Curve for Polylysine Reference: Ranjbar B, Gill P. Circular Dichroism Techniques: Biomolecular and Nanostructural Analyses- A Review. Chemical Biology & Drug Design. 2009;74(2):101-120. Table 1: Features of CD Spectra of Protein Secondary Structures 12

13. Far- UV CD spectra of protein solutions � - sheet turn Random coil  - helix 13 Near- UV CD spectra of protein solutions Kelly, et al. Biochimica et Biophysica Acta 1751 (2005). http://www.slideshare.net/kleppingerb/lipids- and- proteins

14. CD Multivariate SSE Program A calibration model for secondary structure abundance ratios obtained by X- ray crystallography and CD is produced using PCR or PLS, which is then used to estimate the unknown secondary structure of proteins. 14

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