Circular Dichroism Spectroscopy

Circular Dichroism Spectroscopy
Introduction
Amino Acid Structure & Polarity
Protein Structures
Polarization of Light
Circular Dichroism
Measurement of Circular Dichroism
Instrumentation For CD Spectropolarimeter
CD Spectrum
CD Spectra of Protein Secondary Structures
Other - CD Based Techniques
Conclusion
References

1. Prepared by: Aditya Sharma M.S. (Pharm) Pharmaceutical Analysis NIPER Guwahati Roll No. PA/2020-3/049 1 Circular Dichroism Spectroscopy

2. Introduction Amino Acid Structure & Polarity Protein Structures Polarization of Light Circular Dichroism Measurement of Circular Dichroism Instrumentation For CD Spectropolarimeter CD Spectrum CD Spectra of Protein Secondary Structures Other - CD Based Techniques Conclusion References CONTENT 2

3. INTRODUCTION Chiroptical spectroscopy has become one of the most important techniques for the characterization of biomolecules, determination of absolute configuration and stereochemical analysis. 3 Figure 1 : Outline of Circular Dichroism

4. Amino Acid Structure & Polarity 4 N C C H H H R O O H Side Chain Carboxyl Group Amino Group Figure 2: General Structure of Amino Acid

5. Protein Structures Amino acids Alpha helix Beta Pleated Sheet Alpha helix Beta Pleated Sheet 5 Figure 3: Structure of Proteins

6. Polarization of Light x z y y x z Left Handed Right Handed Circularly Polarised Linearly or Plane Polarised Unpolarised Figure 4: Schemes of the electric field components of unpolarized (A) , linearly or plane polarized light (B) . For circularly polarized light (C). A B C 6

7. Optically active molecules absorb left handed and right handed light differentially. This difference in absorbance is called CIRCULAR DICHROISM or CD. Optically active molecules Sample Left Handed Right Handed Circularly Polarised Circular Dichroism 7 Figure 5: Schematic Representation of Circular Dichroism

8. Measurement of Circular Dichroism Wavelength (nm) Wavelength (nm) Dichroism Absorption Circular Dichroism = ΔA = A L – A R ΔA = A L – A R According to Beer Lamberts Law A = εcl ΔA = ε L cl – ε R cl ΔA = Δεcl Δε = ΔA/cl For unit concentration and unit length Δε = ΔA = Circular Dichroism 8

9. Ellipticity Optically active molecules Sample Left Handed Right Handed Circularly Polarised I R I L I R + I L I R - I L θ ΔA = θ/32.982 [θ] = 100 ᳵ θ/(C ᳵ l) Δε = [θ]/3298.2 9 Figure 6 : Schematic Representation of Measurement of Circular Dichroism

10. Instrumentation For CD Spectropolarimeter Figure 7 : I nstrumentation for a common CD spectropolarimeter Monochromator and linear polarizer Detector CD Active Medium PEM High Intensity Light Source PEM = Photo Elastic Modulator PMT = Photomultiplier Tube Xenon lamp Rochon Polarizer PMT 10

11. 11 Figure 8: Advantages of CD Spectropolarimeter

12. 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 Amide, Secondary Structure Aromatic groups, Tertiary structure, Folding Extrinsic groups, Tertiary structure, Ligand binding 12 Figure 9: Schematic Representation of Classification of CD Spectrum Regions

13. CD Spectra of Protein Secondary Structures -ve band (nm) +ve band (nm) α-helix 222 192 208 β-sheet 216 195 Random coil 200 13 Figure 10: Fasman Standard Curve for Polylysine Table 1: Features of CD Spectra of Protein Secondary Structures Reference: Ranjbar B, Gill P. Circular Dichroism Techniques: Biomolecular and Nanostructural Analyses- A Review. Chemical Biology & Drug Design. 2009;74(2):101-120.

14. 14 Figure 11: CD Signal of Protein Depending upon Protein Secondary Structure Reference: Kelly S, Price N. The Use of Circular Dichroism in the Investigation of Protein Structure and Function. Current Protein & Peptide Science. 2000;1(4):349-384.

15. 15 Figure 12: CD Signal of Protein Depending Upon Change in Temperature Reference: Kelly S, Price N. The Use of Circular Dichroism in the Investigation of Protein Structure and Function. Current Protein & Peptide Science. 2000;1(4):349-384.

16. 16 l -1 Figure 13: Chemical structure of the azobenzene cross-linker Figure 14: Models of the cross-linked peptide with the azobenzene group in the trans(left) and cis (right) conformations. Reference: -Kumita J, Smart O, Woolley G. Photo-control of helix content in a short peptide. Proceedings of the National Academy of Sciences. 2000;97(8):3803-3808.

17. Other CD – Based Techniques 17 Figure 15: Other CD – Based Techniques

18. CD is now routinely used to study biological macromolecules (i.e., proteins, nucleic acids, etc.) and also provides useful structural information. R ecent advances have made it possible to improve the time resolution of natural and magnetically induced CD spectral measurements from the millisecond to nanosecond and picosecond time regimes. As an analytical instrument, the technology can determine changes in secondary structure in a qualitative and even semi-quantitative fashion. W ith the easy availability of a wide range of empirical algorithms for secondary structure calculations, new reference databases and other data analysis tools, CD-based techniques should prove to be even more valuable tools in structural biology over the next decades than CD has been in the past 40 or more years since it was first used to estimate biomolecular structures. Conclusion 18

19. Greenfield N. Using circular dichroism spectra to estimate protein secondary structure. Nature Protocols. 2006;1(6):2876-2890. Johnson W. Protein secondary structure and circular dichroism: A practical guide. Proteins: Structure, Function, and Genetics. 1990;7(3):205-214. Matsuo K. Secondary-Structure Analysis of Proteins by Vacuum-Ultraviolet Circular Dichroism Spectroscopy. Journal of Biochemistry. 2004;135(3):405-411. Ranjbar B, Gill P. Circular Dichroism Techniques: Biomolecular and Nanostructural Analyses- A Review. Chemical Biology & Drug Design. 2009;74(2):101-120 . Rogers D, Jasim S, Dyer N, Auvray F, Réfrégiers M, Hirst J. Electronic Circular Dichroism Spectroscopy of Proteins. Chem. 2019;5(11):2751-2774. Kelly S, Jess T, Price N. How to study proteins by circular dichroism. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 2005;1751(2):119-139. Greenfield N. Circular Dichroism (CD) Analyses of Protein-Protein Interactions. Methods in Molecular Biology. 2015;:239-265. Kirkpatrick D, Fain M, Yang J, Trehy M. Enantiomeric impurity analysis using circular dichroism spectroscopy with United States Pharmacopeia liquid chromatographic methods. Journal of Pharmaceutical and Biomedical Analysis. 2018;156:366-371. Kelly S, Price N. The Use of Circular Dichroism in the Investigation of Protein Structure and Function. Current Protein & Peptide Science. 2000;1(4):349-384. Kumita J, Smart O, Woolley G. Photo-control of helix content in a short peptide. Proceedings of the National Academy of Sciences. 2000;97(8):3803-3808. References 19

20. 20 THANK YOU