1. 5P2-1 Chapter 5: Outline Amino Acids Amino acid classesStereoisomers Bioactive AATitration of AA Modified AAAA reactions Peptides Proteins (We are here) Protein structure Fibrous proteins Globular proteins

2. 5P2-2 Protein Function 1.Catalysis 2. Structure 3. Movement 4. Defense 5. Regulation 6. Transport 7. Storage 8. Stress Response

3. 5P2-3 Peptide: a polymer of about 2-100 AAs linked by the peptide (amide) bond. As the amino group and the carboxyl group link, water is lost. PEPTIDES – re-caps

4. 5P2-4 A peptide is written with the N- terminal end to the left and the C- terminal end to the right. H 2 N-Tyr-Ala-Cys-Gly-COOH Name = Tyrosylalanylcysteinylglycine The peptide bond is rigid and planar due to the resonance contribution shown right.

5. 5P2-5 Amino acids consists of carboxyl groups, amino groups, and various R groups can undergo numerous chemical reactions. Polypeptides are linear polymers. They are composed of amino acids that linked together by peptide bonds. Peptides bonds are amide linkages. The linkages formed when the unshared electron pair of the  -amino nitrogen atom of one amino acid attacks the  - carboxyl carbon.

6. 5P2-6 The linked amino acids are referred to as amino acid residues. The amino acid residue with the free amino group is called the N-terminal residue and written to the left of polypeptide. H 2 N-Tyr-Ala-Cys-Gly-COOH Name = Tyrosylalanylcysteinylglycine The amino acid residue with free carboxyl group is called the C-terminal residue and written to the right of a polypeptide.

7. 5P2-7 The C-terminal residues can be identified by a group of enzymes called the carboxypeptidases. The N-terminal residues is determined using Sanger’s method where the polypeptide chain is reacted with 1-fluoro-2,4- dinitrobenzene.

8. 5P2-8 DEFRAGMENTATION OF POLYPEPTIDE # The cleavage or broken up of polypeptides done by using several reagents, mostly the enzymes. # The pancreatic enzyme, trypsin is most commonly used. # Trypsin cleaves peptide bonds on the carboxyl side of either lysine or arginine residues. # Chymotrypsin, another pancreatic enzyme, breaks peptide bonds on the carboxyl side of phenylalanine, tyrosine or trptophan.

9. 5P2-9 # Cyanogen bromide is a reagent specifically cleaves peptide bonds on the carboxyl side of methionine residues. # 1-fluoro-2,4-dinitrobenzene (DNFB) has been used to determine the N-terminal amino acids. # The N-terminal is identified as the dinitrophenyl (DNP) is attached to the terminal after reaction. # Carboxypeptidases hydrolizes peptides one residue at a time from the C-terminal end.

10. 5P2-10 Class discussion page 155 Problem 1 and 2

11. 5P2-11 Proteins by Shape-1 Fibrous proteins exist as long stranded molecules: Eg. Silk, collagen, wool. A collagen segment in space-filling model illustrates this point. Red spheres represent oxygen, grey carbon, and blue nitrogen

12. 5P2-12 Proteins by Shape-2 Globular proteins have somewhat spherical shapes. Most enzymes are globular. Eg. myoglobin, hemoglobin. Myoglobin in space-filling model is the chosen example.

13. 5P2-13 Proteins by Composition Simple Contain only amino acids Conjugated simple protein (apoprotein) prosthetic group (nonprotein) glycoproteins lipoproteins metaloproteins etc. Holo- protein

14. 5P2-14 Four Levels of Protein Structure Primary, 1 o the amino acid sequence Secondary, 2 o 3-D arrangement of backbone atoms in space Tertiary, 3 o 3-D arrangement of all the atoms in space Quaternary, 4 o 3-D arrangement of subunit chains

15. 5P2-15 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.

16. 5P2-16 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

17. 5P2-17 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

18. 5P2-18 Det. Primary Structure: 3b protein Edman’s reagent Phenylthiohydantoin (PTH) derivative of N-term AA Thiazoline derivative

19. 5P2-19 Det. Primary Structure: 4 5. Reassemble peptide fragments from step 3 to give protein. An example follows on the next slide.

20. 5P2-20 Det. Primary Structure: 4b 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 One is C-term Lys is internal !

21. 5P2-21 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:

22. 5P2-22 Secondary Structure The two very important secondary structures of proteins are:  -helix  -pleated sheet Both depend on hydrogen bonding between the amide H and the carbonyl O further down the chain or on a parallel chain.

23. 5P2-23  Helix: Peptide w Hbonds First six C=O to N hydrogen bonds shown

24. 5P2-24  Sheet: stick form Protein G Chain 1 H bonds in dotted red-blue Chain segment 1 Seg 2 Seg 3 Seg 4 H bonds shown in dotted red-blue

25. 5P2-25 B Sheet: Lewis Structure Parallel sheet Antiparallel sheet

26. 5P2-26 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

27. 5P2-27 Supersecondary Structure: 2 Combinations of  helix and  sheet.    meander

28. 5P2-28 Tertiary Structure The configuration of all the atoms in the protein chain: side chains prosthetic groups helical and pleated sheet regions

29. 5P2-29 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

30. 5P2-30 Tertiary interactions: diag. ionic disulfide hydrophobic H bonds or dipole Ion-dipole metal coord’n

31. 5P2-31 Domains Domains are common structural units within the protein that bind an ion or small molecule.

32. 5P2-32 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

33. 5P2-33 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.

34. 5P2-34 Denaturation -loss of protein structure, 2 o  4 o, but not 1 o. 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

35. 5P2-35 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!!

36. 5P2-36 Fibrous Proteins Fibrous proteins have a high concentration of  -helix or  -sheet. Most are structural proteins. Examples include: a-keratin collagen silk fibroin

37. 5P2-37 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.

38. 5P2-38 Roles of Globular proteins - Globular proteins are compact, spherical molecules which are usually water-soluble. - In their functions, usually requires them to bind precisely to other molecules. - Therefore the identity and arrangement of surface amino acids are important. - The acid aminos interact to form a specific binding cavities which are complementary to the structure of a specific molecule, called ligand. - Others is devoted to the binding of regulatory molecules to ensure the maintenance of the polypeptide’s three dimensional structure.

39. 5P2-39 Myoglobin: 2 o and 3 o aspects Globular myoglobin has 153 AA arranged in eight  -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.

40. 5P2-40 Some helical regions Myoglobin: 2 o and 3 o aspects Heme group with iron (orange) at the center

41. 5P2-41 The Heme Group Pyrrole ring N of His F8 binds to fifth site on the iron. His E7 acts as a ”gate” for oxygen.

42. 5P2-42 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.

43. 5P2-43 Hemoglobin A tetrameric protein two  -chains (141 AA) two  -chains (146 AA) four heme units, one in each chain Oxygen binds to heme in hemoglobin cooperatively: as one O 2 is bound, it becomes easier for the next to bind. Lengthy segments of the  and  chains homologous to myoglobin.

44. 5P2-44 Hemoglobin: ribbons + hemes Each chain is in ribbon form and color coded. The heme groups are in space filling form

45. 5P2-45 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.

46. 5P2-46 Differences in structures of Myoglobin and Hemoglobin MyoglobinHemoglobin 1. The protein component of myoglobin, called globin, composed of single polypeptide chain that contains eight sections of  -helix. 2. The oxygen dissociation curve has a hyperbolic shape in a simple pattern. 1. The protein component of haemoglobin, a tetramer, composed of two  -chains and two  – chains. 2. The oxygen dissociation curve has a complicated sigmoidal pattern by the noncovalent interactions among its four subunits.

47. 5P2-47 Continue…. MyoglobinHemoglobin 3. Binds the molecule tightly and release it only when the cell’s oxygen concentration is very low. 4. Has a greater affinity for oxygen, binds more oxygen from blood to muscle. 3. Each  -dimer remain largely unchanged when hemoglobin interconverts between its oxygenated and deoxygenated forms. 4. Has smaller affinity for oxygen, easy to release oxygen to muscle.

48. 5P2-48 Oxygen Binding Curves-2

49. 5P2-49 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 +.

50. 5P2-50 The End Amino Acids, Peptides And Proteins Part 2