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