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Spectroscopy of Proteins

Spectroscopy of Proteins. 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.

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Spectroscopy of Proteins

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  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. 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 Spectroscopic methods

  9. Molecular energy and light spectrum • Emolecule = Eelectronic + Evibrational + Erotational + Espin + Etranslational

  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 Energy levels associated with IR absorbance Molecular vibrations

  13. Derivation and deconvolution

  14. ATR (attenuated total reflectance)-FTIR

  15. Ellipticity: =eL(l)-eR (l) Ellipticity in degrees: Molar Ellipticity: CD Optical activity in proteins • Asymetric atoms ( C of amino acids) • Secondary structures ( helices and  sheets) • Asymetric environment (of aromatic amino acids)

  16. Secondary structure analysis Thermal stability analysis binding analysis

  17. Fluorescence • Excitation • Vibrational losses • 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. Very large molecules Very small molecules unpolarized Lifetime Lifetime Fluorescence Polarization (anisotropy)

  23. Kinetic mechanism of binding

  24. Fluorescence Microscopy

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

  26. X-ray crystallography and NMR

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