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.

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Presentation transcript:

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 E molecule = E electronic + E vibrational + E rotational + E spin + E translational

Absorbance (and transmittance) Beer-Lambert’s law Chromophors in proteins Peptidic bond (UV-CD and FTIR) Aromatic amino acids ( 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  =  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)

Secondary structure analysisThermal 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 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