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Chapter 5: Outline

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. Protein Function. Catalysis 2. Structure 3. Movement 4. Defense

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Chapter 5: Outline

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  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+.

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