Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Protein Structure

1,526 views

Published on

  • Be the first to comment

Protein Structure

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

×