Lecture 9: Protein Characterization

Question 1

Different ways of looking at protein structure

Answer 1

• Infra-red(IR) spectroscopy: Absorption of infrared radiation by peptides is influences by changes in hydrogen bonding from diff secondary structures
• Circular Dichroism(CD) Spectroscopy: Changes in absorbance of circularly polarized UV light from the asymmetry and chirality of amino acids and secondary structurs
• 3D structure techniques: X-ray crystallography, NMR, cryo-EM

Question 2

H-bonding and IR spectra

Answer 2

• Absorbance of amide 1 band can be used to distinguish between alpha helix and beta-sheet
• The stronger the H-bonds, the weaker the C=O bonds
• alpha helix: 1650-1660
• beta sheet: 1620 - 1640
• antiparallel beta sheets: 1675 - 1695
• Random coil: 1640 - 1660

Question 3

Circular Dichroism(CD)

Answer 3

• Chiral alpha carbons and secondary structures preferentially absorb one direction of light over the other
• alpha helix: 222nm, 208 nm(neg), 195nm
• beta sheet: 217 nm(neg)

Question 4

X-ray Crystallography

Answer 4

• Some proteins can form crystals under varying conditions(pH, high salt)
• Crystals placed in an X-ray diffractometer produce diffraction patterns
• The phi and psi angles of each residue define the protein backbone
• 3D structures of proteins can be reconstituted at high resolution

Question 5

Nuclear Magnetic Resonance(NMR)

Answer 5

• NMR based on nuclear spin of certain nuclei, that can be measured in a strong, static magnetic field
• Absorption of electromagnetic radiation can be used to deduce environment of nucleus and determine a proteins structure
• Carried out on proteins in solution
• Can monitor conformational changes, protein folding and interactions with other molecules

Question 6

Cryo-Electron Microscopy

Answer 6

• Larger complexes can be visualized
• Thin layer of protein solution prepared on fine grid and frozen to trap molecules in different orientations
• High powered microscopes measure beam of electrons that pass through protein sample
• Diffraction in beam can be used to determine the structure

Question 7

Protein Chromophores

Answer 7

• Chromophores are functional groups that contain conjugated double bonds that absorb UV light at specific wavelengths
• Aromatic rings and amide carbonyls are important chromophores found in proteins
• Structure of protein influences accessibility of these chromophores to light absorption and characterize protein structure
• Fluorophores absorb and emit light known as fluorescence

Question 8

Tryptophan fluorecense

Answer 8

• Presence of indole ring allows Trp to absorb and emit when excited with UV light
• Trp emiits light between 310-355 nm
• Exact wavelength depends on polarity of environment
• In polar environment, fluorecense is red shifted

Question 9

Fluorescence Microscopy

Answer 9

• Helps see proteins in cellular context
• Fluorescently tagged proteins can show us where certain proteins are in a cell
• Fluorescently tagged antibodies can be used instead of creating fusion proteins
• FRET and BRET can detect distance-dependent protein-protein interactions

Question 10

Fluorescence Based Assays

Answer 10

• FRET: relies on the emission of light from a donor fluorophore which can excite a nearby acceptor fluorophore
• BRET: doesn’t need an external light source for activation. Based on an enzyme-catalyzed reaction that produces light