Protein Structure

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  • 1. Protein Structure www.freelivedoctor.com
  • 2. Why study structure?
    • Structure helps us understand function
    • Many disorders are due to aberrant protein structure
      • Sickle cell anemia
      • Can aid in design of therapeutics
    • Disruption of structure causes disruption of function
      • denaturation
    www.freelivedoctor.com
  • 3. Native Structure
    • The normal structure found in an organism
    • Functional structure
      • Human Insulin
    www.freelivedoctor.com
  • 4. Prosthetic Groups
      • Non-amino acid molecule that is necessary for the protein to function
        • Ex: heme in hemoglobin
        • Mostly an organic molecule
      • Often contain metal ions
    www.freelivedoctor.com
  • 5. Stabilization of Protein Structure
    • H-bonds
      • Backbone
      • Side chain
    • Disulfides
    • Electrostatic
    • Nonpolar forces
    www.freelivedoctor.com
  • 6. Driving of protein folding
    • What provides the energy?
      • Stuff gets moved pretty far
      • Requires energy
      • Driven by entropy increase of solvent
    www.freelivedoctor.com
  • 7. Levels of Protein Structure
    • Primary
    • Secondary
      • Supersecondary
    • Tertiary
    • quaternary
    www.freelivedoctor.com
  • 8. Primary Structure
    • Amino acid sequence
      • Read amino to carboxyl
      • Below is the sequence for insulin
      • malwmrllpl lallalwgpd paaafvnqhl cgshlvealy lvcgergffy tpktrreaed lqvgqvelgg gpgagslqpl alegslqkrg iveqcctsic slyqlenycn
      • Can get sequences from the link below
    www.freelivedoctor.com
  • 9. Determination of primary structure
    • Biochemical
      • Enzymatic digestion
      • Sequential Edman degradation
    • Molecular biological
      • Determine nucleotide sequence
      • Deduce amino acid sequence
    www.freelivedoctor.com
  • 10. Determination of primary structure
    • Determine amino acid content
      • Boil protein in 6 M HCl
    • Protein cleavage
      • Use enzymes that cut at different places
        • Chymotrypsin: cuts at C-end of aromatic aa
        • Trypsin: cuts at C-end of + charged aa
        • Cyanogen bromide: cuts after M
      • Determine sequences of peptides
      • Line up
    www.freelivedoctor.com
  • 11. Primary Sequence determination example
    • Trypsin Digests:
    • N-T-W-M, D-T-W-M-I-K, G-Y-M-Q-F-V-L-G-M-S-R
    • CNBr digests:
    • Q-F-V-L-G-M, D-T-W-M, S-R-N-T-W-M, I-K-G-Y-M
          • What is the correct sequence?
    www.freelivedoctor.com
  • 12. Secondary structure
    • Simple folding
      • α -helix
      • β -pleated sheet
      • Collagen helix
    www.freelivedoctor.com
  • 13. α -helix
    • Right handed helix
    • 3.6 aa/ turn
    • Backbone H-bonding between carboxyl oxygen and amino nitrogen 4 aa away
      • C=O on aa 1 bonds to N-H on 5
      • C=O on aa 2 bonds to N-H on 6 etc
    • Proline doesn’t fit into helix
    www.freelivedoctor.com
  • 14. α -helix
    • R groups perpendicular to helix
    • To right is view down helix axis
    • AA close in primary sequence of protein not necessarily close in final structure
    • AA far away in primary often close in final structure
    www.freelivedoctor.com
  • 15. Beta sheet
    • Parallel or antiparallel sheet
    • Backbone H-bonding between C=O and N-H on adjacent strands
    • Sheet is pleated
    www.freelivedoctor.com
  • 16. Beta Sheet side view
    • R- groups below and above plane of sheet
    • R-groups on adjacent aa very far apart
    www.freelivedoctor.com
  • 17. Collagen helix
    • Triple helix
    • Helices wrapped around each other.
    • Sequence is G-X-P-G-X-P etc
    • Found in tendons and ligaments
    • Very strong
    • H-bonding between strands
    www.freelivedoctor.com
  • 18. Supersecondary structure
    • Structural motifs
    • Repeating patterns of secondary structures
      • Beta barrels
      • Alpha-beta-alpha-beta
      • Examples in next slides
    www.freelivedoctor.com
  • 19. Helix- turn- helix www.freelivedoctor.com
  • 20. Beta sandwiches www.freelivedoctor.com
  • 21. 4 alpha bundle www.freelivedoctor.com
  • 22. Beta Barrel www.freelivedoctor.com
  • 23. Alpha beta horseshoe www.freelivedoctor.com
  • 24. Tertiary Structure
    • Final 3-D structure of the protein
    • Folded native form
    • aa close together in primary sequence often far apart in tertiary
    • aa far apart in primary may be close in tertiary
    www.freelivedoctor.com
  • 25. Myoglobin structure www.freelivedoctor.com
  • 26. Quaternary Structure
    • Subunits
    • Made from more than one polypeptide chain
    • Held together by weak interactions
    www.freelivedoctor.com
  • 27. Hemoglobin www.freelivedoctor.com
  • 28. Protein Structure Determination
    • X-ray crystallography
      • X-rays diffract off of molecule
      • Pattern is Fourier transform of actual structure
      • 2 angstrom resolution
      • Have to make crystal
    • NMR
      • Similar to MRI technology
      • Can do in solution
      • Resolution not as good
    www.freelivedoctor.com
  • 29. Protein purification
    • Start with source
      • Cells from animal or plant
      • Molecular bio
    • Grind up
    • Centrifuge
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  • 30. Protein purification
    • Chromatography
      • Ion exchange
      • Size exclusion
      • Affinity chromatography
        • Use of antibodies
    www.freelivedoctor.com
  • 31. Protein Purification
    • Assays
      • Functional
        • Activity
          • Define
      • Protein amount
        • Colorimetric
        • Beer’s law
        • ELISA
      • Determination of specific activity
        • Activity / mg protein
    www.freelivedoctor.com
  • 32. SDS PAGE
    • Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis
    • Separates by size
    • Distance travelled is function of log(MW)
    • Native v denaturing
    www.freelivedoctor.com