spectroscopy of proteins. proteins the final product of the genes, translated form genes (mutation...
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![Page 1: 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](https://reader035.vdocuments.us/reader035/viewer/2022062320/56649d7e5503460f94a61db9/html5/thumbnails/1.jpg)
Spectroscopy of Proteins
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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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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
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Levels of protein structure
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The α-helix and β-sheet
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Protein spectroscopy- what for?
• Structural analysis- Shape, size and form- secondary and tertiary conforamtions
• quantification
• Interaction with other molecules (proteins, ligands and solutes).
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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
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Molecular energy and light spectrum
• Emolecule = Eelectronic + Evibrational + Erotational + Espin + Etranslational
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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)
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Absorbance of aromatic amino acids
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FTIR
Molecular vibrations
Energy levels associated with IR absorbance
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Derivation and deconvolution
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ATR (attenuated total reflectance)-FTIR
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CD
=LR 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)
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Secondary structure analysis Thermal stability analysis binding analysis
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Fluorescence
1. Excitation
2. Vibrational losses
3. Emission
Fluorimetric setup
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Probes used in biology
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GFP –Green Fluorescence Protein
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Tryptophan fluorescence
Trp blue shift
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Fluorescence Resonance Energy Transfer (FRET)
Energy at excited state of the donor is transmitted to an acceptor
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Fluorescence Polarization (anisotropy)
Very large molecules
Lifetime Lifetime
unpolarized
Very small molecules
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Kinetic mechanism of binding
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Fluorescence Microscopy
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Light scattering
Dynamic light scatteringSmall angle X-ray scattering
Solution versus crystal
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X-ray crystallography and NMR