CHEMISTRY OF PEPTIDES AND PROTEINS

1. CHEMISTRYOF PEPTIDES AND PROTEINS Dr. VinithaRamanathPai Professor, Dept of Biochemistry, Yenepoya Medical College.

2. Exercise for revision of “Chemistry of Amino acids” Fill in the blank with the most appropriate answer

3. Exercise : ______is an example for a hormone synthesized from amino acids. In allergic reactions the ___________ that is produced is from the amino acid ____________. The source of D - amino acids in the body is _______. AnswEr 1) Thyroid hormones / catecholamines Answer 2) Histamine; Histidine Answer 3) Bacterial cell wall/ Antibiotics

4. Exercise : 4. An amino acid with pI of 4.6 is put into a buffer of pH 8.0 . The charge on the amino acid is __________. All amino acids except glycine have at least one ___________________. Answer 4) Negative (Anion) Answer 5 ) Asymmetric atom

5. CHEMISTRY OF PEPTIDES

6. Formation of peptide bond + Amino acid 1 Amino acid 2 R2 R2 O C - OH H H α α + + α α C C C C NH2 NH2 H N H H N COOH COOH R1 R1 H H H2O Condensation reaction R2 R2 O C O C H H Dipeptide α α α α C C C C NH2 NH2 N H COOH COOH R1 R1 H H Peptide bond

7. Characters of a peptide bond……. O C O C Dipeptide H H R2 R2 α α α α N H C C C C NH2 NH2 COOH COOH Amino terminal / N terminal C terminal / Carboxy terminal R1 R1 H H • Peptide bond : Covalent linkage - Amide linkage. Free  - COOH group One end has a free  NH2 group • Made up of C=O and N-H groups – “Planar”,”trans” • Two amino acids are linked by one peptide bond. • CN bond has partial double bond character – does not rotate easily. • In general , ‘n’ no. of amino acids are linked by ‘n-1’ no. of peptide bonds. • The α –C, carbonyl C and amideNforms the backbone of the peptide. • The side chains R1, R2, etc., projectfrom the backbone of the peptide.

8. Polypeptides, Proteins……… + + Amino acid 1 Amino acid 2 Amino acid 3 R2 R2 Tripeptide O C O C H H H H N α α α α α α C O C C C C C C COOH NH2 NH2 N H N H R1 R1 H H R3 Tetrapeptide : linear chain of 4 amino acids linked by 3 peptide bonds Oligopeptide : linear chain of 2-10 amino acids Polypeptide : linear chain of 10- 50 amino acids Protein : linear chain of  50 amino acids.

9. Polypeptides, Proteins……… O C O C H H R2 H N H α α α α α α N H C C C C C C COOH NH2 NH2 C O R1 R1 H R3 Each amino acid in the peptide is called a residue Sequence of a peptide .... NH2- Gly – Asp - Phe – Val – Lys - COOH 1 2 3 4 5 NH2-Glycyl - Aspartyl - Phenyl alanyl - Valyl – Lysine-COOH • Pentapeptide - Name from N- terminal to C- terminal. • Replace “ine” of every amino acid with “yl” except the “C” terminal residue.

10. Peptide Examples Function Dipeptides Carnosine Anserine Skeletal muscle chelate and enhance copper uptake. Tripeptide Glutathione γ Glu- Cys - Gly Thyrotropin releasing hormone (TRH) Hypothalamic hormone Novapeptides Oxytocin Vasopressin Bradykinin uterine contractions ↑ blood pressure & has anti diuretic action. vasodilator causes contraction of smooth muscle.

11. Other Peptide hormones……. Glucagon - 29 amino acids increases blood sugar ACTH - 39 amino acids Pituitary hormone Gastrin - 34 Acts on stomach Angiotensin I - 10 regulates BP Enkephalins - 5 Released in the brain Insulin - 51 amino acids Lowers blood sugar

12. Exercise : A peptide has 12 amino acids. How many N – terminal ends does it have ? Glutathione is tripeptide. Name the amino acids The back bone of the peptide is made up of _______ Atoms. Answer 1 ) One Answer 2 ) γ Glu- Cys - Gly Answer 3)α –C, carbonyl C, amideN

13. CHEMISTRY OF PROTEINS

14. Contents – Definition – General Aspects – Classification – Structural Organization of Proteins – Structures of Insulin, Hemoglobin and Collagen – Denaturation of Proteins – Isoelectric pH of Proteins – Water-solubility of Proteins

15. Introduction to Protein Chemistry Definition Proteins are linear, heteropolymers of L-  - amino acids, that arelinked by peptide bonds and containing more than 50 amino acids.

16. Introduction to Protein Chemistry General aspects Proteinscontain • Primary / standard - L,  - amino acids (20 in number), • Occasional secondary amino acids (post- translationally modified amino acids). • Majority of proteins are composed of 100 to 4000 amino acid residues. • The amino acids are linked by peptide bonds.

17. Structure of a Protein Free - amino group Free - carboxyl group Backbone of the protein A protein chain has direction. Conventionally, the direction is from N terminal to C terminal (N→C). ‘R’ groups – projecting out of the backbone

18. Introduction to Protein Chemistry General aspects • The peptide bonds are part of the backbone of the protein chain. • The R-groups (side chains) project out from the backbone on either side. • Terminal ends of the protein : has a free • –NH2 group at one end, called amino terminal or N- terminal and a free COOH group at the other end, called carboxy terminal or C- terminal.

19. Native Conformation of a protein A protein molecule is a linear chain of amino acids. (Biologically inactive) spontaneously folds into a three-dimensional structure/conformation Biologically active Native protein (3 dimensional) Native protein

20. Structure of Proteins is important because.. • Every protein is made up of the 20 different L, α - amino acids. • The number and sequence of these amino acids is different in different proteins. • Each protein has a unique structure which is three dimensional- Native structure / conformation of the protein. • Conformation gives structure to the protein. • The function of the protein is dependent on its 3D structure.

21. Subunits in a protein • Proteins are made up of one, or sometimes more than one polypeptide chain/protein chain. • one chain are referred to as monomeric proteins. E.g., Myoglobin • more than one polypeptide chain are referred to as oligomeric proteins. E.g., Hemoglobin Subunit

22. Classification of proteins Based on biological function Based on Chemical composition Based on shape Based on no. of subunits

23. I. Classification based on biolgoical function of protein Class (10) Examples Catalytic proteins Enzymes Transport proteins Hb, Transferrin Membrane Transport proteins Na+ - K+ ATPase Storage proteins Myoglobin, Ferritin Contractile proteins Actin, Myosin Structural proteins Collagen, Keratin Protective proteins Immunoglobulins, / Defence proteins Regulatory proteins Hormones Clotting proteins Clotting factors Receptor proteins Hormone receptors

24. I. Classification of Proteins based on Chemical composition 3 classes Simple proteins Derived proteins Conjugated proteins Made up of only amino acids Derived from simple and conjugated proteins Made up of Protein part + non-protein part e.g., Plasma albumin; histones e.g., Gelatin, peptones, proteoses e.g., egg albumin; Casein, Hemoglobin 2 sub-classes 7 sub-classes 6 sub-classes

25. Sub-classification of Simple Proteins ( 7 subclasses) Sub-class Examples Plasma albumin Albumins Globulins Plasma globulin; Ovaglobulin Glutelin of wheat ; Oryzenin of rice Glutelins Gliadin of wheat ; Zein of corn Prolamines Nucleoproteins ; Globin of Hb Histones Nucleoproteins of sperm Protamines Collagen of bone; Keratin of hair, nail, horn, hoof Sclero-proteins

26. Sub-classification of Conjugated Proteins Protein + Non Protein Conjugated Proteins Apo - protein + prosthetic group Conjugated Proteins Examples for prosthetic group: carbohydrate, lipid, nucleic acids , phosphate, coloured groups, metal ions. Sub-classification of conjugated proteins based on the prosthetic group - 6 subclasses

27. Sub-classification of Conjugated Proteins Conjugated protein Non protein part Examples Histones, Protamines Nucleoproteins Nucleic acids Glycoproteins/ Proteoglycans / Mucoproteins Mucin, Immunoglobulins Hormones Carbohydrates (4%) Colored prosthetic group Heme in Hb Riboflavin in Flavoproteins Chromoproteins Phosphoproteins Casein in milk Vitellin in egg Phosphate VLDL, LDL, HDL, Chylomicrons Lipid Lipoproteins Metalloproteins Metal ions Hb – Fe2+ Ceruloplasmin Cu+

28. Sub-classification of Derived Protein Definition : Derived from simple and conjugated proteins. 2 Sub classes Primary derived protein Denatured protein Secondary derived Partially hydrolysed protein No cleavage of peptide bonds Cleavage of peptide bonds No decrease in MW Decrease in MW Change in the structure of the protein Protein becomes smaller

29. Protein Protean Metaprotein (coagulated protein) 1° derived protein –denatured protein by action of denaturing agents. No change in MW Protein Proteoses Peptones Peptides Amino acids 2° derived protein –Decrease in MW Progressive hydrolysis of peptide bond.

30. Structure of the answer for Classification of Proteins based on Chemical composition Simple proteins Derived proteins Conjugated proteins Albumins Primary derived Globulins Nucleoproteins Glutelins Secondary derived Glycoproteins Prolamines Chromoproteins Histones Protamines Lipoproteins Sclero-proteins Metalloproteins 3 classes Definition Definition Definition Subclasses Subclasses Subclasses Phosphoproteins Definition & examples of the subclasses

31. III. Classification based on Molecular shape of protein (2 classes) Axial ratio = Length of the Protein Breadth of the protein Fibrous protein Globular protein Shape : Spherical / Ovoid Elongated Mol. Wt : Low High Solubility : soluble Less soluble Axial ratio : less than 10 More than 10 Eg : Albumin, globulin, enzymes, histones Collagen, Keratin, Elastin

32. Exercise for revision Match the following

33. Plant proteins •  - NH2 group • Tripeptide • Secondary derived protein • Metalloprotein • Nucleoproteins • Primary derived protein • Amide linkage • Conjugated protein • Match the following • Gelatin • Transferrin • Histone • Metaproteins • Glutelins • Peptide bond • N – terminal • TRH (d) (e) (f) (g) (a) (h) (b) (c)

34. Exercise for revision Fill in the blank with the most appropriate answer

35. Excercise : Albumin from serum was mixed with pepsin and incubated at 37C for 30 minutes. What are the products Which class do the products belong to ? 2. Hemoglobin belongs to __________ class , and ______ subclass of proteins. 3. The three dimensional structure of a protein is called as ___________________. Answer 1) Proteoses, peptones, peptides - derived protein Answer 2) Conjugated proteins; chromoproteins Answer 3) Native conformation

36. Exercise : 4. The axial ratio of a protein is 150. It is a _____________protein. Creatinephosphokinase is an enzyme. It has two subunits. It is a ______________ protein. Answer 4) Fibrous Answer 5 ) oligomeric

37. Structure of Proteins Proteins have 4 different levels of structural organisation: Primary 1° Secondary 2° Tertiary 3° Quaternary 4° HIGHER LEVELS OF STRUCTURAL ORGANIZATION

38. The Bonds responsible for Protein structure: 2 types Non covalent bonds Covalent bonds Hydrogen bond Peptide bonds Disulfide bonds Hydrophobic bond/ interaction Electrostatic/ ionic/ salt bond or bridge /interaction Vander Waals interaction Covalent Bonds : 1. Peptide Bonds - discussed

39. Covalent Bonds : 2. Disulfide bridge formation Cystiene Cystiene H H α α COOH C C HS H2C CH2SH COOH NH2 NH2 2H Removal of hydrogens dehydrogenation H H α α COOH C C COOH S H2C CH2S NH2 NH2 Disulphide bridge Cystine Diamino Dicarboxylic amino acid

40. Disulfide bridge Intra-chain disulfide bridge. Both the cystienes are in the same polypeptide chain, It folds the molecules Inter-chain disulfide bridge The two cystienes are on different polypeptide chains It holds the chains together

41. Non Covalent Bonds Hydrogen bond : -Bond formed by sharing a single H atom between two electronegative atoms, i.e., ‘N’ and ‘O’ atoms of peptide bonds. Hydrogen bond • Electrostatic / ionic / salt bond : • Interaction between oppositely charged groups, • NH3+--------------------OOC on (i) terminal ends (ii) the R groups.

42. Hydrophobic bond / interaction : Interaction between non polar side chains of amino acids on the same protein molecule Vander Waals interaction : Weak short distance forces either attractive or repulsive in nature.

43. Exercise : Identify the bonds 1. Non – polar / hydrophobic force of interaction 2. Hydrogen bond 3. Ionic bond

44. Describe this protein Primary Structure of Insulin 2 polypeptide chains – 51 residues A Chain – 21 amino acid residues B Chain – 30 amino acid residues Intrachain – A Cys 6 - A Cys11 3 Disulfide bridges Interchain – A Cys7 - B Cys 7 A Cys19 - B Cys 20

45. Primary structure of Proteins Peptide bond is responsible for the primary structure of protein. Importance of Primary structure of Proteins: Total number of amino acids Sequence of amino acids N- terminal and C- terminal residues Position of Cys residues which form disulfide bridge Primary structure of Insulin : Elucidated by Sanger in 1955 – Awarded Nobel Prize in 1958.

46. Structure of Insulin 1 1 A B S 6 7 7 S S S 11 19 S S 21 20 30 Thr 2 polypeptide chains – 51 residues A Chain – 21 amino acid residues B Chain – 30 amino acid residues 3 Disulfide bridges Intrachain – A Cys 6 - A Cys11 Interchain – A Cys7 - B Cys 7 A Cys19 - B Cys 20 • Porcine (pig) insulin is closest to human insulin B30 is Ala (porcine) instead of Thr (in human insulin)

47. Secondary structure of Protein H H R2 H R4 H R6 H N O C H N O C H N O C α α α α α α α α α α α α C C C O NH2 C C C C C C C C C C C O C O C N H N H N H R3 R1 H R3 H R5 H 1 2 3 4 5 6 7 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 folding and twisting 1 6 2 Hydrogen bond Hydrogen bond results in folding and twisting

48. Secondary structure of Proteins: Definition : configurational relationship of residues which are about 3-4 amino acids apart in the linear primary sequence. Stabilized by : hydrogen bonds between the H of the NH group and O of C= O of two different peptide bonds. which are 3-4 amino acid residues away in the same polypeptide chain. This gives rise to folding and twisting of the primary structure into secondary structure.

49. Regular folding & twisting of the polypeptide chain brought about by hydrogen bonding is called Secondary structure of protein. Types of secondary structures • α Helix • β pleated sheet • β bend / β turn • Loop region • Disordered region/ random coil • Triple helix

50. α Helix First described by the Scientists Pauling (Nobel Prize 1954) and Corey in 1951. • is spiral rod like structure • Backbone is in a Right handed helix • Stabilised by Hydrogen bonds • NH and C=O which are 3.6 amino acid residues away • H bonds are parallel to the central imaginary axis.