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