Protein Structure

1,310 views
1,167 views

Published on

0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
1,310
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
52
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

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

×