1. Chapter 24 Amino Acids and Proteins Created By Prof. Gary F. Porter, Ph.D. Copyright © 2014 by John Wiley & Sons, Inc. All rights reserved.

2. Table of Contents 1.IntroductionIntroduction 2.Amino AcidsAmino Acids 3.Synthesis of α–Amino AcidsSynthesis of α–Amino Acids 4.Polypeptides and ProteinsPolypeptides and Proteins 5.Primary Structure of Polypeptides and ProteinsPrimary Structure of Polypeptides and Proteins 6. Examples of Polypeptide and Protein Primary Structure Examples of Polypeptide and Protein Primary Structure 7. Polypeptide and Protein SynthesisPolypeptide and Protein Synthesis 8. Secondary, Tertiary, and Quaternary Structures of ProteinsSecondary, Tertiary, and Quaternary Structures of Proteins 9. Introduction to EnzymesIntroduction to Enzymes 10. Purification and Analysis of Polypeptides and ProteinsPurification and Analysis of Polypeptides and Proteins 11. ProteomicsProteomics © 2014 by John Wiley & Sons, Inc. All rights reserved.

3. Amino acids are the monomeric unit for proteins. Proteins are polyamides. There are: – 20 different common α-amino acids, and – 4 levels of protein structure. 1. Introduction © 2014 by John Wiley & Sons, Inc. All rights reserved.

4. In the 20 common amino acids, the α carbon has an L configuration. 1. Introduction © 2014 by John Wiley & Sons, Inc. All rights reserved.

5. 2A.Structures and Names: The four classes of amino acids are: Neutral, Nonpolar Neutral, Polar Charged Positive, Polar Acidic (at pH 7) Charged Negative, Polar Basic (at pH 7) 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

6. 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

7. 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

8. 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

9. 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

10. 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

11. 2B. Essential Amino Acids: – Are not synthesized by higher animals. – Also, Histidine and arginine are essential in infants. The Essential Eight ValineTryptophan LeucineThreonine IsoleucineMethionine PhenylalanineLysine 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

12. 2C. Amino Acids as Dipolar Ions: – Contain a basic group (-NH 2 ) and an acidic group (-CO 2 H), and each of these has a pKa. – Exist as dipolar ions at physiological pH. Dipolar ions are also called zwitterions. Isoelectric point (pI) is the pH at which the net charge on an amino acid is 0. 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

13. Alanine has two ionization equilibria: 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

14. Note the titration curve for Alanine below. – As base is added and pH is increased, the net charge on Alanine goes from positive to negative. 2. Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

15. 3A. Method 1: From Potassium Phthalimide. Methionine Synthesis 3. Synthesis of α–Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

16. 3B. Method 2: The Strecker Synthesis treat aldehyde with NH 3 and HCN, and produce an α amino acid. 3. Synthesis of α–Amino Acids © 2014 by John Wiley & Sons, Inc. All rights reserved.

17. Peptides – are amino acids joined together to form a molecule. Peptide Bonds – are amide linkages between the amino acids. N terminus on left C terminus on right No. of Amino Acids linked together Name 2Dipeptide 3Tripeptide 3-10Oligopeptide Multiple polypeptidesProtein 4. Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

18. 4A. Hydrolysis Peptide bond cleavage occurs in 6 M HCl for 24 h. AAs separate on cation-exchange resin. – AAs will bind at low pH. Amino Acids (AA) – AAs release as pH increases. Resin 4. Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

19. Detection of AAs: – Ninhydrin Reagent – λ max = 570nm Formation of blue complex occurs with this reagent. 4. Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

20. Primary Structure is defined by: – amino acid sequence, – molecular weight, – amino acid composition. Edman Degradation: – identification of N-Terminus, – sequential degradation of AAs from N- terminus, – Automated sequencing is good up to ~60 AAs. 5.Primary Structure of Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

21. 5A. Edman Degradation: uses phenylisothiocyanate, cleaves the N-terminal AA, as a phenylthiohydantion, The R group identifies the AA. 5.Primary Structure of Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

22. 5B. Sanger N-Terminus Analysis: an N-terminal AA analysis, uses 2,4-dinitrofluorobenzene. 5.Primary Structure of Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

23. 5C. C-Terminus Analysis – uses carboxypeptidases, – continually cleaves carboxyl AAs, and – can only be used with smaller peptide chains. 5.Primary Structure of Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

24. 6B. Insulin – is secreted by pancreas, and – regulates (lowers) blood glucose. Thousands of other polypeptides and protein structures are known. 6.Examples of Polypeptide and Protein Primary Structure © 2014 by John Wiley & Sons, Inc. All rights reserved.

25. 7D. Automated Peptide Synthesis – Involves solid phase addition, – addition of AA, – followed by wash. – High yield at each step is realized. FMOC = Fluorenylmethyloxy carbonyl 7. Polypeptide and Protein Synthesis © 2014 by John Wiley & Sons, Inc. All rights reserved.

26. Secondary Structure is defined by conformations of polypeptides. Specific types are – α helix, – β sheets, and – coil or loop. Rotation about the α-carbon atom in each residue. – AA residues have limited or no rotation. – The side groups have free rotation. – There is free rotation about the α-carbon but not the peptide bond. 8. Secondary, Tertiary, and Quaternary Structures of Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

27. Secondary Structure β pleated sheet (below) has – high nonpolar AA content, and – predominates in silk. α helix (right) has – 3.6 AA’s per right handed turn, and – is in fibrous proteins. 8. Secondary, Tertiary, and Quaternary Structures of Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

28. 8B. Tertiary Structure – has a three dimensional shape that arises from secondary structures orientation to each other, and – results in two classes of proteins. Fibrous Proteins have mainly α helices. Globular Proteins – have all types of secondary structure. – AA classes have specific locations: nonpolar are favored in the interior and polar aare favored on the exterior. 8. Secondary, Tertiary, and Quaternary Structures of Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

29. Carbonic Anhydrase Myoglobin 8. Secondary, Tertiary, and Quaternary Structures of Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

30. 8C. Quaternary Structures Three dimensional shape that arises from polypeptide chain orientation to each other. See the four polypeptide chains of hemoglobin to the right. 8. Secondary, Tertiary, and Quaternary Structures of Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

31. Enzymes – catalyze all chemical reactions found in cellular metabolism, – have highly specific reactants called substrates, – form an enzyme-substrate complex, and – are stereospecific. Enzyme-substrate complex – induces conformational changes in the enzyme, – is the place where the reaction is catalyzed, and – resides within the active site. 9. Introduction to Enzymes © 2014 by John Wiley & Sons, Inc. All rights reserved.

32. Competitive Inhibitors – have a similar structure to substrate and – binds to the active site instead of the substrate. Cofactors – is an extra molecule required for enzyme activity. – i.e. metal ions Coenzyme – Organic cofactor 9. Introduction to Enzymes © 2014 by John Wiley & Sons, Inc. All rights reserved.

33. Lysozymes cleave the cell wall of gram positive bacteria. Lysozymes hydrolyze the glycosidic linkages in peptoglycans. Blue AAs are found at the active site. 10. Lysozyme: Mode of Action of an Enzyme © 2014 by John Wiley & Sons, Inc. All rights reserved.

34. Pancreatic digestive enzymes Chymotrypsin – is activated from Chymotrypsinogen – is a peptidase – Catalytic triad - Asp 102, His 57, Ser 195 – The triad only forms in Chymotrypsin – The inactive form exists in the pancreas to avoid damage 11. Serine Proteases © 2014 by John Wiley & Sons, Inc. All rights reserved.

35. Hemoglobin is a conjugated protein with a nonprotein prosthetic group. Heme is the prosthetic group. Fe 2+ 12. Hemoglobin: A Conjugated Protein © 2014 by John Wiley & Sons, Inc. All rights reserved.

36. 13A. Purification – Involves chemical and chromatographic means. – Depends upon molecular weight pI, and stability. 13. Purification and Analysis of Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

37. 13B. Analysis Methods – Gel Electrophoresis (based upon pI) – Mass Spectroscopy Obtains molecular weight – Electrospray Ionization (ESI) uses peak corresponds to m/z ratio and mixtures separated by HPLC – Matrix-assisted Laser Desorption Ionization (MALDI) Used for ionization of nonvolatile molecules 13. Purification and Analysis of Polypeptides and Proteins © 2014 by John Wiley & Sons, Inc. All rights reserved.

38. Involves the identification, structure, and function of proteins 14. Proteomics © 2014 by John Wiley & Sons, Inc. All rights reserved.

39. Ch. 21 - 39  END OF CHAPTER 24 