Structure and Functions of Proteins in Microbiology

1. B.Sc (HONOURS) MICROBIOLOGY (CBCS STRUCTURE) CC-3: BIOCHEMISTRY (THEORY) SEMESTER –2 MCB-A-CC-2-3-TH TOTAL HOURS: 50 CREDITS: 4 Unit 4 Proteins No. of Hours: 10 Functions of proteins, Primary structures of proteins: Amino acids, the building blocks of proteins. General formula of amino acid and concept of zwitterion. Titration curve of amino acid and its Significance, Classification, biochemical structure and notation of standard protein amino acids Ninhydrinreaction.Natural modifications of amino acids in proteins hydrolysine, cystine and hydroxyproline, Non protein amino acids: Gramicidin, beta-alanine, D-alanine and D- glutamic acid Oligopeptides: Structure and functions of naturally occurring glutathione and insulin and synthetic aspartame, Secondary structure of proteins: Peptide unit and its salient features. The alpha helix, the beta pleated sheet and their occurrence in proteins, Tertiary and quaternary structures of proteins. Forces holding the polypeptide together. Human haemoglobin structure, Quaternary structures of proteins

2. R Amino Acids • Amino Acids are the building units of proteins. Proteins are polymers of amino acids linked together by “ Peptide bond” • There are about 300 amino acids occur in nature. Only 20 of them occur in proteins. • Structure of amino acids: • Each amino acid has 4 different groups attached to α- carbon (which is C-atom next to COOH). These 4 groups are : amino group, COOH gp, • Hydrogen atom and side • Chain ( R group)

3. Amino Acids • While their name implies that amino acids are compounds that contain an —NH2 group and a —COOH group, these groups are actually present as —NH3+ and —COO– respectively. • They are classified as a, b, g, etc. amino acids according the carbon that bears the nitrogen.

4. The 20 (22) Key Amino Acids • More than 700 amino acids occur naturally, but 20 (22)of them are especially important. • These 22 amino acids are the building blocks of proteins. • They take part in translation • All are a-amino acids. • They differ in respect to the (R) group attached to the a carbon.

7. Selenocysteine Selenocysteine is the 21st amino acid that naturally occur in proteins in all three domains of life, but not in every lineage. Selenocysteine is a cysteine analogue and acts as a building block of selenoproteins. Selenocysteine is present in several enzymes.

8. Pyrrolysine Pyrrolysine, the 22nd amino acid, encoded by the 'amber’ stop  codon (UAG) is an  amino acid that is used in the biosynthesis of proteins in some  methanogenic archea and eubacteria. Pyrrolysine is not present in humans

9. Redefinition of Codons

10. Amino acids have both basic and acidic groups and so can act as base or acid. Neutral amino acids (monobasic, monocarboxylic) exist in aqueous solution as “ Zwitter ion” i.e. contain both positive and negative charge. Zwitter ion is electrically neutral and can’t migrate into electric field. Isoelectric point (IEP) It is the pH at which the zwitter ion is formed. e.g IEP of alanine is 6 Amphoteric properties of amino acids

11. At physiological PH (7.4), -COOH gp is dissociated forming a negatively charged carboxylate ion (COO-) and amino gp is protonated forming positively charged ion (NH3+) forming Zwitter ion • N.B. Prolineis an imino acid not amino acid • Classification of amino acids

12. O O •• •• •• •• + – •• •• •• H3NCH2C O H2NCH2C OH •• •• •• Properties of Glycine solubility: soluble in water; not soluble in nonpolar solvent more consistent with this than this

13. O •• •• + – •• H3NCH2C O •• •• Properties of Glycine more consistent with this called a zwitterion or dipolar ion

14. O •• •• + •• H3NCH2C OH •• Acid-Base Properties of Glycine • The zwitterionic structure of glycine also follows from considering its acid-base properties. • A good way to think about this is to start with the structure of glycine in strongly acidic solution, say pH = 1. • At pH = 1, glycine exists in its protonated form (a monocation).

15. O •• •• + •• H3NCH2C OH •• Acid-Base Properties of Glycine • As the pH is raised, which is the first proton to be removed? Is it the proton attached to the positively charged nitrogen, or is it the proton of the carboxyl group? • One can choose between them by estimating their respective pKas. typical ammonium ion: pKa ~9 typical carboxylic acid: pKa ~5

16. O •• •• + •• H3NCH2C OH •• Acid-Base Properties of Glycine • The more acidic proton belongs to the COOH group. It is the first one removed as the pH is raised. typical carboxylic acid: pKa ~5

17. O •• •• + – •• H3NCH2C O •• •• O •• •• + •• H3NCH2C OH •• Acid-Base Properties of Glycine • Therefore, the more stable neutral form of glycine is the zwitterion. typical carboxylic acid: pKa ~5

18. O •• •• + •• H3NCH2C OH •• Acid-Base Properties of Glycine • The measured pKa of glycine is 2.34. • Glycine is stronger than a typical carboxylic acid because the positively charged N acts as an electron-withdrawing, acid-strengthening substituent on the a carbon. typical carboxylic acid: pKa ~5

19. O O •• •• •• •• – + HO – – •• •• •• H3NCH2C O H2NCH2C O •• •• •• •• Acid-Base Properties of Glycine A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased further. • The pKa for removal of this proton is 9.60. This value is about the same as that for NH4+

20. O •• •• + •• H3NCH2C OH •• O •• •• + – •• H3NCH2C O •• •• O •• •• – •• •• H2NCH2C O •• •• Isoelectric Point pI • The pH at which the concentration of the zwitterion is a maximum is called the isoelectric point. • Its numerical value is the average of the two pKas. • The pI of glycine is 5.97. pKa = 2.34 pKa = 9.60

21. Titration curve of amino acids One -NH2 group One –COOH group

22. One -NH2 group One –COOH group

26. Physical Properties of Amino Acid 1. Solubility: Most of the amino acids are usually soluble in water, and insoluble in organic solvents. 2. Melting Point: Amino acids are generally melt at higher temperature of ten above 2000C. 3. Taste: Amino acids may be sweet (Gly, Ala & Val), tasteless (Leu) or Bitter (Arg & Ile). 4. Optical Properties: All amino acids possess optical isomers due to the presence of asymmetric α-carbon atoms.

27. Chemical Properties of Amino Acid Chemical reactions of amino acids due to carboxylandamino groups: Reaction due to carboxyl groups Reaction due to amino groups Reaction due to carboxyl and amino groups

28. Chemical Properties of Amino Acid Chemical reactions of amino acids due to carboxylandamino groups: Reaction due to carboxyl groups Reaction due to amino groups a) Reaction with Formaldehyde a) Decarboxylation: b) Reaction with Benzaldehyde b) Reaction with Alkalies (Salt formation): c) Reaction with Nitrous acid (Van slyke reaction): c) Reaction with Alcohols (Esterification) : d) Reaction with Sanger’s Reagent d) Reaction with Amines: e) Reaction with DANSYL Chloride f) Reaction with acylating agents (Acylation):

29. Reaction due to carboxyl groups

30. Reaction due to carboxyl groups

31. Reaction due to carboxyl groups

32. Reaction due to carboxyl groups

33. Reaction due to amino groups

34. Reaction due to amino groups

35. Reaction due to amino groups

36. Reaction due to amino groups

37. Reaction due to amino groups

38. Reaction due to amino groups

39. Reaction due to amino groups

40. Reaction due to carboxyl and amino groups

43. Neutral aromatic amino acids a-Phenyl alanine : It’s alanine in which one hydrogen of CH3 is substituted with phenyl group. So it’s called phenyl alanine b- Tyrosine: - it is P- hydroxy phenyl alanine - it is classified as phenolic amino acid c- Tryptophan: as it contains indole ring so it is classified as heterocyclic amino acid

44. Neutral heterocyclic amino acids a- Tryptophan: contains indole ring b- Proline: In proline, amino group enters in the ring formation being α-imino gp so proline is an α-imino acid rather than α-amino acid

45. Acidic Amino acids at physiological pH will carry negative charge. e.g. Aspartic acid (aspartate) and Glutamic acid (glutamate). Aspargine and Glutamine:They are amide forms of aspartate and glutamate in which side chain COOH groups are amidated. They are classified as neutral amino acids.

47. Classification according to polarity of side chain (R): Polar amino acids: in which R contains polar hydrophilic group so can forms hydrogen bond with H2O. In those amino acids, R may contain: 1- OH group : as in serine, threonine and tyrosine 2- SH group : as in cysteine 3- amide group: as in glutamine and aspargine 4- NH2 group or nitrogen act as a base (basic amino acids ): as lysine, arginine and histidine 5- COOH group ( acidic amino acids): as aspartate and glutamate .

48. Classification according to polarity of side chain (R) Non polar amino acids R is alkyl hydrophobic group which can’t enter in hydrogen bond formation. 9 amino acids are non polar ( glycine, alanine, valine, leucine, isoleucine, phenyl alanine, tryptophan, proline and methionine)

49. Peptide bond formation α-carboxyl group of one amino acid (with side chain R1) forms a covalent peptide bond with α-amino group of another amino acid ( with the side chain R2) by removal of a molecule of water. The result is : Dipeptide ( i.e. Two amino acids linked by one peptide bond). By the same way, the dipeptide can then forms a second peptide bond with a third amino acid (with side chain R3) to give Tripeptide. Repetition of this process generates a polypeptide or protein of specific amino acid sequence.

50. Peptide bond formation: - Each polypeptide chain starts on the left side by free amino group of the first amino acid enter in chain formation . It is termed (N- terminus). - Each polypeptide chain ends on the right side by free COOH group of the last amino acid and termed (C-terminus).