PROTEINS & ENZYMES

1. PROTEINS & ENZYMES

2. PROTEINS BIOLOGICAL ROLES Structure - collagen (skin), keratin (hair) Transport - membrane transporters Enzymes - catalysts Motility - myosin Immune system - Ig Metabolic regulation - hormones

3. DNA mRNA PROTEIN DNA mRNA Peptide/protein AA AA AA AA……..

4. Structure of AAs - L amino acids Different types of amino acids - R group Amino acid modifications Ionisation of AAs Peptide bond Biologically important peptides

5. AMINO ACID STRUCTURE -carbon H H2N C COOH R amino group carboxyl group Side chain

6. L-amino acids make up proteins mirror plane H HOOC C R H H2N C COOH R NH2 D-amino acid L- amino acid Enantiomers

7. Side chain (R ) determines AA properties HYDROPHOBIC (and aliphatic) R= (non-polar) H CH2 CH CH3 CH3 CH CH3 CH3 CH3 Glycine (G) Alanine (A) Valine (V) Leucine (L) Also Isoleucine (I), methionine (M), proline (P)

8. HYDROPHOBIC (and aromatic) R= (non-polar) CH2 C CH NH CH2 Phenylalanine (F) Tryptophan (W) Also: Tyrosin (Y)

9. HYDROPHILIC(and charged) R= (polar) CH2 CH2 CH2 CH2 NH3 CH2 CH2 C O O CH2 C O O Aspartate (D) + Glutamate (E) Lysine (K) Also Arginine (R ) and histidine (H) +ve charge

10. HYDROPHILIC (and neutral) R= (polar) CH2 CH2 C O NH2 CH2 OH CH2 C O NH2 Serine (S) Asparagine (N) Glutamine (Q) Also Threonine (T) and Cysteine (C)

11. Amino Acid properties HYDROPHILIC R groups - H bonding with water In aqueous environment on OUTSIDE of protein HYDROPHOBIC R groups -Hydrophobic interactions In aqueous environment on INSIDE of protein

12. PROTEIN IN AQUEOUS ENVIRONMENT hydrophilic hydrophobic

13. Special Features of some Amino acids Methionine - 1st amino acid in protein sequence (N-terminal) Cysteine (SH grp) - disulphide bond formation Aromatic amino acids (tryptophan, tyrosine, phenylalanine) UV absorption at 280nm

14. AMINO ACID MODIFICATION Serine and threonine (OH grp) -O-linked glycosylation (glycoprotein) - reversible phosphorylation (ser/thr kinases) Asparagine (NH2 grp) - N-linked glycosylation Proline (ring) - forces bend in protein structure - can be hydroxylated(e.g. in collagen) 20 amino acids coded, many more from modification

15. IONISATION STATE OF AMINO ACIDS H H3N C COOH H H H3N C COO H H H2N C COO H + + pH 1 + charge pH 7 no net charge pH 11 - charge

16. H H3N C COO H ISOELECTRIC POINT pI pH when no net charge + pI=7 Aspartate H H3N C COO CH2 COO H H3N C COO CH2 COOH + + pH 7 net -ve charge pH 4 no net charge (pI)

17. R group ionisation ACIDIC Glutamate, aspartate - R grp COO- at neutral pH pI < 7 BASIC Lysine, arginine - R group NH3+ at neutral pH pI > 7

18. PEPTIDE BOND FORMATION R1 NH2 CH COOH R2 NH2 CH COOH + R1 H2N CH C R2 NH CH COOH O + H2O Peptide bond Dipeptide

19. PEPTIDE UNIT IS RIGID Peptide unit R1 H2N CH C R2 N CH COOH O H Partial double bond character Rotational freedom

20. PROTEIN 3D STRUCTURE RIGID PEPTIDE BOND - well defined structure ROTATIONAL FREEDOM - proteins can fold in many ways

21. POLYPEPTIDE CHAIN Dipeptide tripeptide polypeptide R1 NH2 CH C R2 NH CH C O R3 NH CH C R4 NH CH COOH O O Amino terminal residue Carboxyl terminal residue Protein sequence from amino to carboxyl end N C

22. NATURAL PROTEINS Between 50 - 2 000 amino acid residues Mean molecular mass of amino acid residue = 100 Daltons Mean molecular mass of protein = 5 000 to 200 000 Daltons = 5 to 200 kiloDaltons (kDa)

23. 3D STRUCTURE OF PROTEINS PEPTIDES POLYPEPTIDES PROTEINS Increasing complexity of structure 4 different levels of protein structure

24. 1y structure AMINO ACID SEQUENCE Only interaction btw AAs is peptide bond No other interactions or stabilising bonds

25. 2Y STRUCTURE Local structure of polypeptide chain H BONDING between carbonyl oxygen and amide hydrogen R1 NH2 CH C R2 N CH C O R3 N CH C R4 N CH C O O O H H H O O R6 O R5 R7 C CH N C CH N C CH N H H H

26. 2 TYPES OF 2y STRUCTURE Alpha helix (-helix) Rod-like structure Coiled peptide chain R groups extend outwards from axis H bonding btwn CO and NH 4 residues away 3.6 amino acids per turn of helix

27. Model of -helix H bonds CO HN

28. - helix in proteins myoglobin and haemoglobin 75% alpha-helix

29.  -helical coiled coils 2 or more -helices entwined - very strong eg Myosin, tropomyosin in muscle Fibrin in blood clots Keratin in hair SUPERHELIX

30. 2. Beta sheets (-sheets) Polypeptide chain is extended H bonding between CO and NH on different polypeptide strands Strands can run in same direction (parallel -sheet) or in opposite direction (anti-parallel -sheet)

31. Model of -sheet Anti-parallel SILK - anti-parallel -sheets Strong structure

32. Special helix in collagen Rod shaped structure - NOT same as -helix More open than -helix 3 amino acid residues per turn Conserved sequence Gly-X-Y-Gly-X-Y- (X and Y are proline and hydroxyproline)

33. Collagen triple helix Each strand forms a helix 3 collagen strands entwine - triple helix Glycine occupies every 3rd position - small, fits in interior ( Gly-X-Y-Gly-X-Y-Gly-X-Y……) Proline and hydroxyproline to exterior

34. PROTEIN 3y STRUCTURE Side chain (R group) interactions Most are weak, non-covalent interactions - but stabilise protein Spatial arrangement and interactions of AA’s far apart Responsible for folded, biologically active protein

35. 3D STRUCTURE Of myoglobin

36. TYPES OF BONDING H bonding (ser, thr OH groups) Electrostatic interactions between charged groups (lys, arg, his, glu, asp) Hydrophobic interactions (leu, val, phe….etc) Van der Waals’ forces (short range) Disulphide bonding between cysteine residues Strong interaction

37. 3y structure bonds

38. PROTEIN DENATURATION DISRUPTS BONDING insoluble Heat - breaks weak bonding (eg egg white) pH -affects H bonding and electrostatic interactions Detergents and organic solvents(hydrophobic bonds) Chaotropic agents eg urea, guanidine HCl Form H bonds with AAs and disrupt existing H bonding and hydrophobic interactions

39. 4y STRUCTURE Complex of 2 or more separate polypeptide chains Interactions between subunits Non-covalent and covalent interactions

40. MODEL of HAEMOGLOBIN 4 subunits interacting Has 4y structure

41. SUMMARY OF PROTEIN STRUCTURE 1Y 3Y 4Y 2Y Tetrameric protein

42. Importance of protein structure

43. 3D STRUCTURE IMPORTANT FOR PROTEIN FUNCTION Allows protein to interact with other molecules Form bonds with other molecules Usually weak but specific interactions Why proteins act as enzymes, receptors etc

44. IMPORTANCE OF 1y STRUCTURE Denaturing agents: Urea, guanidine HCl - disrupt H and hydrophobic bonds Mercaptoethanol - reducing agent Reduces disulphide bonds (-S-S-- SH ) - SH

45. Denaturation of 3y structure Denatured protein

46. Denaturation of 3y structure can be reversed Remove urea and mercaptoethanol Denatured protein

47. CONCLUSION All information for complex 3D structure contained in amino acid sequence 1y sequence dictates structure Chemically, folding slow process Physiologically, protein folding involves chaperone proteins - scaffold, help folding

48. IMPORTANCE OF CORRECT 1Y STRUCTURE GENE MUTATIONS Changes in AA sequence SUBSTITUTIONS - conservative - same type of AA - radical - different type AA Change can affect ligand binding or enzyme activity Can affect shape of protein (esp cysteine substitutions)

49. INSERTIONS OR DELETIONS Can cause loss of protein function But not always - if remove non-essential part Deletions/substitutions - can determine binding site residues

50. PROTEIN ENVIRONMENT IN AQUEOUS ENVIRONMENT e.g. water soluble protein HYDROPHILIC AMINO ACIDS OUTSIDE (H bonding, electrostatic interactions) HYDROPHOBIC AMINO ACIDS INSIDE (hydrophobic interactions, Van der Waals’ forces)