Amino acid and Protein Chemistry

1. Amino acid and Protein Chemistry Dr.S.Chakravarty MBBS,MD

2. Learningobjectives At the end of this lecture a student should be able to :- • Classify the amino acids based on polarity, nutritional requirements, side chains and chemical side chains • Describe the pathways of Protein turnover. Explain Ubiquitin proteosome pathway • State the four levels of protein structure and explain how the sequence of amino acids leads to the final three dimensional configuration of the protein • Explain Protein misfolding and its clinical implications • Analyse the Concept of zwitter ions and its clinical applications

3. - COO + a H N 3 H L-Form Amino Acid Structure Carboxylic group Amino group H = Glycine R group CH3 = Alanine

4. STEREOISOMERISM Compounds having same structural formula , but differ in spatial configuration are known as stereoisomerism. Penultimate Carbon

5. Mirror Images of Amino Acid a a Mirror image Enantiomers

7. Amino Acids Classification • Based on polarity • Based on side chain • Based on metabolic function • Based on nutritional requirement

8. Based on Side chain

9. Aliphatic side chains (non-polar)

10. Imino acid ( Non-polar)

11. Glycine : • Smallest amino acid • Inhibitory neurotransmitter • Most common amino acid of collagen • Valine, Leucine and isoleucine: • Leucine – ketogenic amino acid 2. Maple syrup urine disease • Proline – imino acid , disrupts amino acid structure

12. Aromatic amino acids Indole ring

13. Phenyl Alanine: • Essential amino acid : forms tyrosine • Def : phenyl ketonuria ( phenyl alanine hydroxylase deficiency) • Tyrosine : • Precursor of melanin • Catecholamines – dopamine, norepinephrine and epinephrine which act as neurotransmitters • Parkinson disease : def of dopamine • synthesis of thyroxine . • Tryptophan : (indole ring) • Melatonin - circadian rhytm • Serotonin – mood alterations • Source of Niacin – def causes hartnup’s disease

14. Sulphur containing amino acids

15. Cysteine : • Used to make disulfide bonds – secondary structure of proteins • Glutathione – detoxifying agent and free radical scavenger. • Pharmacology : N –acetyl cysteine • Paracetamol toxicity • Cystic fibrosis • Cyclophosphamide toxicity – treatment • Methionine : • S-adenosylMethionine : methyl donor • Homocysteine – derived from methionine

16. Side chains with Basic groups(polar ) Guanidino group Imidazole group

17. Lysine and arginine– due to positive charges , main components of histone proteins. • Arginine : • Synthesis of nitric oxide • Release of urea • Semi-essential amino acid – positive nitrogen balance • Histidine : • Histamine – major inflammatory mediator .

18. Acidic groups and their amides(polar)

19. Side chains with –OH groups

20. 21st and 22ndaminoacid • Selenocysteine • Pyrrolysine

21. Based on Nutritional requirement Essential Amino Acids • Not synthesized in the body • Essential in diet Non - Essential Amino Acids • Synthesized in the body Nutritionally essential mnemonic – MATTVILPHLY OR PVT TIM HaLL

22. Classification based on essentiality PVT TIM HaLL Arginine also.

23. Based on Metabolic function

24. Classification of Amino Acids by Polarity Neutral Acidic Basic Asp Asn Arg Ser POLAR Cys Tyr His Gln Thr Lys Glu Ala Gly Ile Phe Trp NON- POLAR Val Leu Pro Met Non-polar substances likely to be inside the cell membrane , polar things OUTSIDE !

25. Zwitter ion • An amino acid acts as a zwitterion, i.e., it can be either: • Positively charged in an acid solution • Negatively charged in an alkaline solution • Neutral at the isoelectric point.

26. cation anion

27. Formation of Peptide Bond

28. 1 COOH COOH NH2 NH2 2 O 2 1 NH2 CN COOH H Formation of Peptide Bonds by Dehydration Amino acids are connected head to tail Dehydration -H2O Carbodiimide

29. Amino acids are joined by peptide bonds

30. Characteristics of PEPTIDE BOND • Partial double bond(The distance is 1.32 A which is midway b/w single bond (1.49 A)and double bond(1.27A)) • Rigid and Planar • The partial double bond renders the amide group planar, occurring in either the cis or trans isomers. •  In the unfolded state of proteins, the peptide groups are free to isomerize and adopt both isomers; however, in the folded state, only a single isomer is adopted at each position (with rare exceptions).

33. Numbering of amino acids in Peptides and Proteins • Free alpha amino group at one end – AMINO TERMINAL( N –TERMINAL ) • The amino acid contributing the alpha amino group is called as first amino acid. • Biosynthesis of protein starts from this terminal end only. • Free carboxy group on other end – CARBOXY TERMINAL(C –TERMNAL ) –> LAST AMINO ACID

34. Amino acid sequences

35. PROTEINS • Greek Proteiosmeaning PRIMARY • BUILDING BLOCKS OF THE BODY • 3/4TH DRY BODY WEIGHT = PROTEINS • CONTAIN C, H , O, N as major components, while S and P are minor constituents. • N content of ordinary proteins is 16% by weight.

36. PROTEOMICS Proteomics is the large scale study of proteins , particularly their structures , functions and interactions.

37. Human Genome Project Completed in 2003, all genes in Human DNA have been identified……so……….

38. WHY PROTEOMICS ??

39. WHY PROTEOMICS ? • Average no of genes is 40,000- 60, 000, average no . of proteins is more than ½ a million in humans. • Average no of proteins per gene -- one to two in bacteria, three in yeast three to six per gene ,in human beings . • In addition to genetic makeup , many other factors determine proteins behavior and expression- factors like pH, oxygen depletion , chemicals and drugs etc.

40. WHY PROTEOMICS ? • The 3D structure of proteins are being discovered by techniques such as X-ray crystallography and NMR . • This has led to Precise predictions of catalytic mechanisms Protein - protein associations Protein nucleic acid interactions GREATER INSIGHT INTO BIOLOGICAL FUNCTIONS

41. WHY PROTEOMICS ? • Functions of proteins are modulated at many points from transcription to post-translational levels which cannot be predicted from the analysis of nucleic acids alone.DNA can be the information archive but how the information is used is another matter! • Not all diseases have a Genetic basis …… • Poor correlation between abundance of m RNA transcribed from DNA , and the number of respected proteins translated from m RNA. • Insight and better understanding into many clinical problems, new tools for diagnosis , new drug-development

42. THE NEXT STEP AFTER GENOMICS….

43. PEPTIDES • Proteins are made by polymerisation of amino acids through peptide bonds. • 2 AA joined to form a DIPEPTIDE • 3 AA -> TRIPEPTIDE • 4 AA-> TERTAPEPTIDE • 5- 10 AA -> OLIGOPEPTIDE • 10- 50 POLYPEPTIDE • >50 PROTEIN 3 AA means 20 3 COMBINATIONS = 8000 possible types of peptides. An ordinary protein with 100 amino acid = 20100 possible combinations !!!

44. BIOLOGICALLY IMPORTANT PEPTIDES

45. Biology/Chemistry of Protein Structure Primary Secondary Tertiary Quaternary Assembly Folding Packing Interaction S T R U C T U R E P R O C E S S

46. PRIMARY STRUCTURE OF PROTEINS The sequence of the amino acids in a polypeptide chain denotes its primary structure. • linear • ordered • 1 dimensional • sequence of amino acid polymer • by convention, written from amino end to carboxyl end • a perfectly linear amino acid polymer is neither functional nor energetically favorable  folding!

47. primary structure of human insulin CHAIN 1: GIVEQ CCTSI CSLYQ LENYC N CHAIN 2: FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT

48. Proteins & Peptides Must Be Purified Prior to Analysis. This is achieved by various methods like :- • Column Chromatography • Partition Chromatography • Size exclusion chromatography • Absorption Chromatography • Ion-Exchange Chromatography • Affinity Chromatography • Peptides Are Purified by Reversed-Phase High-Pressure Chromatography • Protein Purity Is Assessed by iso electric focussing and Polyacrylamide Gel Electrophoresis (PAGE)

49. PROTEOMIC TOOLS - 2D GEL ELECTROPHORESIS • 1st DIMENSION – SEPARATION BASED ON CHARGE - ISO ELECTRIC FOCUSING • 2nd DIMENSION – SEPARATION BASED ON MOLECULAR MASS – PAGE

50. IEF pH1 2 3 4 5 6 7 8 9 10 11 12 13 14 MW Protein mixture P AGE Visualization of approx 1000 to 3000 proteins per gel COMPARE WITH PREVIOUS RECORDS