Chapter 5: Outline

1. Chapter 5: Outline • Amino Acids • Amino acid classes Stereoisomers • Bioactive AA Titration of AA • Modified AA AA reactions • Peptides • Proteins (We are here) • Protein structure • Fibrous proteins • Globular proteins

2. Protein Function • Catalysis • 2. Structure • 3. Movement • 4. Defense • 5. Regulation • 6. Transport • 7. Storage • 8. Stress Response

3. Proteins by Shape-1 • Fibrous proteins exist as long stranded molecules: Eg. Silk, collagen, wool. A collagen segment in space-filling mode illustrates this point. Red spheres represent oxygen, grey carbon, and blue nitrogen

4. Proteins by Shape-2 • Globular proteins have somewhat spherical shapes. Most enzymes are globular. Eg. myoglobin, hemoglobin. Myoglobin in space-filling mode is the chosen example.

5. Proteins by Composition Holo- protein • Simple • Contain only amino acids • Conjugated • simple protein (apoprotein) • prostetic group (nonprotein) • glycoproteins • lipoproteins • metaloproteins • etc.

6. Four Levels of Protein Structure • Primary, 1o • the amino acid sequence • Secondary, 2o • 3-D arrangement of backbone atoms in space • Tertiary, 3o • 3-D arrangement of all the atoms in space • Quaternary, 4o • 3-D arrangement of subunit chains

7. Determining Primary Structure • 1. Hydrolyze protein with hot 6M HCl. • Identify AA and % of each. • Usually done by chromatography • 2. Identify the N-term and C-term AAs • C-term via carboxypeptidase • N-term via Sanger’s Reagent, DNFB • 2,4-dinitrofluorobenzene • Often step 2 can be skipped today.

8. Det. Primary Structure: 2 • 3. Selectively fragment large proteins into smaller ones. • Eg. Tripsin: cleave to leave Arg or Lys as C-term AA • Eg. Chymotrypsin: cleave to leave Tyr or Trp or Phe as C-term AA • Eg. Cyanogen bromide cleaves at internal Met leaving Met as C-term homoserine lactone

9. Det. Primary Structure: 3 • 4. Determine AA sequence of peptides with AA sequencer using Edman’s reagent: • phenyl isothiocyanate which reacts with the N-term AA • See the next slide

10. Det. Primary Structure: 3b Thiazoline derivative protein Edman’s reagent Phenylthiohydantoin (PTH) derivative of N-term AA

11. Det. Primary Structure: 4 • 5. Reassemble peptide fragments from step 3 to give protein. • An example follows on the next slide.

12. Det. Primary Structure: 4b One is C-term • A twelve AA peptide was hydrolyzed. • Trypsin hydrolysis: • Leu-Ser-Tyr-Gly-Ile-Arg • Thr-Ala-Met-Phe-Val-Lys • Chymotrypsin hydrolysis • Val-Lys-Leu-Ser-Tyr • Gly-Ile-Arg • Thr-Ala-Met-Phe • Deduce the AA sequence Lys is internal!

13. Det. Primary Structure: 4c • Tr Leu-Ser-Tyr-Gly-Ile-Arg • Ct Gly-Ile-Arg • Ct Val-Lys-Leu-Ser-Tyr • Tr Thr-Ala-Met-Phe-Val-Lys • Ct Thr-Ala-Met-Phe • The complete sequence is: • Thr-Ala-Met-Phe-Val-Lys-Leu-Ser-Tyr-Gly-Ile-Arg Keeping in mind the N-term AA and overlaping the sequences properly gives:

14. Secondary Structure • The two very important secondary structures of proteins are: • a-helix • b-pleated sheet • Both depend on hydrogen bonding between the amide H and the carbonyl O further down the chain or on a parallel chain.

15. a Helix: Peptide w Hbonds First six C=O to N hydrogen bonds shown

16. b Sheet: stick form Protein G H bonds shown in dotted red-blue H bonds in dotted red-blue Chain segment 1 Seg 2 Seg 3 Chain 1 Seg 4

17. B Sheet: Lewis Structure Antiparallel sheet Parallel sheet

18. Supersecondary Structure • Reverse turns in a protein chain allow helices and sheets to align side-by-side • Common AA found at turns are: • glycine: small size allows a turn • proline: geometry favors a turn

19. Supersecondary Structure: 2 bab b meander aa Combinations of a helix and b sheet.

20. Tertiary Structure • The configuration of all the atoms in the protein chain: • side chains • prosthetic groups • helical and pleated sheet regions

21. Tertiary Structure: 2 • Protein folding attractions: • 1. Noncovalent forces • a. Inter and intrachain H bonding • b. Hydrophobic interactions • c. Electrostatic attractions • + to - ionic attraction • d. Complexation with metal ions • e. Ion-dipole • 2. Covalent disulfide bridges

22. Tertiary interactions: diag. disulfide ionic H bonds or dipole hydrophobic Ion-dipole metal coord’n

23. Domains • Domains are common structural units within the protein that bind an ion or small molecule.

24. Quaternary Structure-1 • Quaternary structure is the result of noncovalent interactions between two or more protein chains. • Oligomers are multisubunit proteins with all or some identical subunits. • The subunits are called protomers. • two subunits are called dimers • four subunits are called tetramers

25. Quaternary Structure-2 • If a change in structure on one chain causes changes in structure at another site, the protein is said to be allosteric. • Many enzymes exhibit allosteric control features. • Hemoglobin is a classic example of an allosteric protein.

26. Denaturation • -loss of protein structure, 2o 4o, but not 1o. • 1. Strong acid or base • 2. Organic solvents • 3. Detergents • 4. Reducing agents • 5. Salt concentrations • 6. Heavy metal ions • 7. Temperature changes • 8. Mechanical stress

27. Denaturation-2 • Denaturing destroys the physiological function of the protein. • Function may be restored if the correct conditions for the protein function are restored. • But! Cooling a hardboiled egg does not restore protein function!!

28. Fibrous Proteins • Fibrous proteins have a high concentration of a-helix or b-sheet. Most are structural proteins. • Examples include: • a-keratin • collagen • silk fibroin

29. Globular Proteins • Usually bind substrates within a hydrophobic cleft in the structure. • Myoglobin and hemoglobin are typical examples of globular proteins. • Both are hemoproteins and each is involved in oxygen metabolism.

30. Myoglobin: 2o and 3o aspects • Globular myoglobin has 153 AA arranged in eight a-helical regions labeled A-H. • The prosthetic heme group is necessary for its function, oxygen storage in mammalian muscle tissue. • His E7 and F8 are important for locating the heme group within the protein and for binding oxygen. • A representation of myoglobin follows with the helical regions shown as ribbons.

31. Myoglobin: 2o and 3o aspects Some helical regions Heme group with iron (orange) at the center

32. The Heme Group Pyrrole ring N of His F8 binds to fifth site on the iron. His E7 acts as a ”gate” for oxygen.

33. Binding Site for Heme • Lower His bonds covalently to iron(II) • Oxygen coordinates to sixth site on iron and the upper His acts as a “gate” for the oxygen.

34. Hemoglobin • A tetrameric protein • two a-chains (141 AA) • two b-chains (146 AA) • four heme units, one in each chain • Oxygen binds to heme in hemoglobin cooperatively: as one O2 is bound, it becomes easier for the next to bind. • Lengthy segments of the a and b chains homologous to myoglobin.

35. Hemoglobin: ribbons + hemes • Each chain is in ribbon form and color coded. • The heme groups are in space filling form

36. Oxygen Binding Curves • Oxygen bonds differently to hemoglobin and myoglobin. • Myoglobin shows normal behavior while hemoglobinn shows cooperative behavior. Each oxygen added to a heme makes additon of the next one easier. • The myoglobin curve is hyperbolic. • The hemoglobin curve is sigmoidal.

37. Oxygen Binding Curves-2

38. The Bohr Effect (H+ and Hb) • Lungs: • pH higher than in actively metabolizing tissue. (Low H+). Hb binds oxygen and releases H+. • Muscle at Work: • pH lower (H+ product of metabolism). Hb releases oxygen and binds H+.