1. Spectroscopy of Proteins

2. Proteins The final product of the genes, translated form genes (mutation in gene leads to a mutated protein) Made of a verity of 20 amino acid building blocks Exert all the biological functions of the organism: enzymes, antibodies, cytoskeletons, hormones, receptors

3. Protein characteristics Unbranched polymer Folds into an accurate three dimensional structure (globular structure) Correct folding is essential for the protein to exert its functions- tight structure-function relationship

4. Levels of protein structure

5. amino acid and peptide bond

6. The α-helix and β-sheet

7. Protein spectroscopy- what for? Structural analysis- Shape, size and form- secondary and tertiary conforamtions quantification Interaction with other molecules (proteins, ligands and solutes).

8. Spectroscopic methods Absorbance- UV-vis, FTIR Circular Dichroism (CD) Fluorescence- internal, labeling, polarization Light scattering- DLS, SAXS NMR X-ray diffraction (crystallography) Resolution of Structural analysis methods Low: UV-vis absorbance, DLS, fluorescence Medium: FTIR, CD, SAXS High: X-ray diffraction, NMR

9. Molecular energy and light spectrum E molecule = E electronic + E vibrational + E rotational + E spin + E translational

10. Absorbance (and transmittance) Beer-Lambert’s law Chromophors in proteins Peptidic bond (UV-CD and FTIR) Aromatic amino acids (260-300 nm) Attached probe (varies, mostly vis)

11. Absorbance of aromatic amino acids

12. FTIR Molecular vibrations Energy levels associated with IR absorbance

13. Derivation and deconvolution

14. ATR (attenuated total reflectance)- FTIR

15. CD  =  L  R  Ellipticity: Molar Ellipticity: Ellipticity in degrees: Optical activity in proteins Asymetric atoms ( C  of amino acids) Secondary structures (  helices and  sheets) Asymetric environment (of aromatic amino acids)

16. Secondary structure analysisThermal stability analysis binding analysis

17. Fluorescence 1.Excitation 2.Vibrational losses 3.Emission Fluorimetric setup

18. Probes used in biology

19. GFP –Green Fluorescence Protein

20. Tryptophan fluorescence Trp blue shift

21. Fluorescence Resonance Energy Transfer (FRET) Energy at excited state of the donor is transmitted to an acceptor

22. Fluorescence Polarization (anisotropy) Very large molecules Lifetime unpolarized Very small molecules

23. Kinetic mechanism of binding

24. Fluorescence Microscopy

25. Light scattering Dynamic light scatteringSmall angle X-ray scattering Solution versus crystal

26. X-ray crystallography and NMR