The document discusses various aspects of protein structure, including: 1) Proteins have primary, secondary, tertiary, and quaternary levels of structure that determine their shape and function. 2) Secondary structures like alpha helices and beta sheets involve hydrogen bonding between amino acids in the protein chain. 3) Tertiary structure refers to a protein's final 3D shape after folding. Quaternary structure involves interactions between protein subunits. 4) Techniques like X-ray crystallography and NMR spectroscopy can be used to determine protein structures at high resolutions.

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 www.freelivedoctor.com

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