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 formsecondary and tertiary conforamtions
• quantification
• Interaction with other molecules (proteins,
ligands and solutes).
Spectroscopic methods
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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
Energy levels associated
with IR absorbance
Molecular vibrations
Derivation and deconvolution
ATR (attenuated total reflectance)FTIR
CD
Ellipticity:
=L(l)-R (l)
Ellipticity in degrees:
Molar Ellipticity:
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
Fluorimetric setup
1.
Excitation
2.
Vibrational losses
3.
Emission
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
Very small molecules
unpolarized
Lifetime
Lifetime
Kinetic mechanism of binding
Fluorescence Microscopy
Light scattering
Small angle X-ray scattering
Solution versus crystal
Dynamic light scattering
X-ray crystallography and NMR