Peptides and proteins structure and functions

Structure and functions of peptides and proteins for undergraduate students

1. Peptide s and Proteins: Structure and Functions R.C . Gupta Professor and Head Dept. of Biochemistry National Institute of Medical Sciences Jaipur, India

2. EM B - R C G Peptides and proteins are polymers of amino acids Their structure and functions depend upon: Nature of amino acids present in them Sequence of amino acids Spatial relationship of amino acids

3. E M B - R C G Peptides are relatively small polymers Generally, polymer s having less than 100 amino acids are known as peptides; those with 100 or more are known as proteins Many peptides are formed from breakdown of proteins Peptides

4. E M B - R C G EMB-RCG

5. E M B - R C G SH | HOOC — CH —CH—CH —C—N—CH—C—N—CH —COOH 2 2 2 NH | 2 CH | 2 O || O || H | H | Glutathione (reduced) Glutathione Glutathione is a tripeptide ( g - glutamyl - cysteinyl -glycine) EMB-RCG

6. E M B - R C G

7. E M B - R C G Glutathione is required for: Detoxification of H 2 O 2 , fatty acid peroxides and some xenobiotics Catalytic activity of many enzymes

8. E M B - R C G Bradykinin is formed in plasma from an a 2 -globulin It is formed by the proteolytic action of trypsin or some enzymes present in snake venom It is a nonapeptide : Arg –Pro–Pro– Gly – Phe – Ser –Pro– Phe – Arg Bradykinin

9. E M B - R C G Increase in the permeability of capillaries Bradykinin causes: Vasodilatation Broncho-constriction

10. E M B - R C G Angiotensin

11. E M B - R C G

12. E M B - R C G Renin ACE Angiotensin I Angiotensin II Angiotensinogen

13. Raises blood pressure Increases the force of contraction of heart Causes vasoconstriction Angiotensin II:

14. Vasopressin Cys -Tyr- Phe - Gln - Asn - Cys -Pro- Arg - Gly

15. Oxytocin Cys -Tyr-Ile- Gln - Asn - Cys -Pro- Leu - Gly

16. Thyrotropin-releasing hormone (TRH ) Pyroglutamate – Histidine – Proline

17. Met- enkephalin Tyr- Gly - Gly - Phe -Met

18. E M B - R C G Proteins Large polymers of amino acids Have complex structures Perform important functions in living organisms

19. E M B - R C G Some general functions performed by proteins in our body are: Maintenance of pH of body fluids Maintenance of osmotic pressure of plasma and intracellular fluid

20. E M B - R C G Classification EMB-RCG

21. E M B - R C G EMB-RCG

22. E M B - R C G EMB-RCG

23. E M B - R C G EMB-RCG

24. E M B - R C G Globulins Soluble in dilute salt solutions but are insoluble in water Heat- coagulable Precipitated on half-saturation with ammonium sulphate Examples are ovoglobulin , lactoglobulin , and serum globulin EMB-RCG

25. E M B - R C G EMB-RCG

26. E M B - R C G EMB-RCG

27. E M B - R C G EMB-RCG

28. E M B - R C G EMB-RCG

29. E M B - R C G EMB-RCG

30. E M B - R C G EMB-RCG

31. E M B - R C G EMB-RCG

32. E M B - R C G EMB-RCG

33. E M B - R C G EMB-RCG

34. E M B - R C G EMB-RCG

35. E M B - R C G EMB-RCG

36. E M B - R C G EMB-RCG

37. E M B - R C G EMB-RCG

38. E M B - R C G EMB-RCG

39. E M B - R C G EMB-RCG

40. E M B - R C G Secondary derived proteins Formed by hydrolysis of native proteins Include primary proteoses , secondary proteoses and peptones in the decreasing order of size EMB-RCG

41. Proteins perform a variety of functions Functions are closely related to the structures of proteins Fundamentally, all proteins are made of amino acids linked to one another by peptide bonds Structural organization of proteins

42. E M B - R C G A complex three-dimensional structure is formed by: The three-dimensional structure is also known as conformation of the protein Union of several peptide chains with one another Coiling and folding of peptide chains

43. E M B - R C G EMB-RCG Non-covalent or weak bonds Structure of proteins is formed by: Covalent or strong bonds

44. E M B - R C G EMB-RCG These include: 1. Peptide bonds 2. Disulphide bonds Covalent Bonds These bonds are relatively strong

45. E M B - R C G Peptide bonds

46. Peptide bond ׀ EMB-RCG

47. Disulphide bonds

48. — HN CH CO — — | CH 2 | SH — HN CH CO — — | CH 2 | S | S | CH 2 | — HN — CH — CO — SH | CH 2 | — HN — CH — CO — A cysteine residue Disulphide bond between two cysteine residues EMB-RCG — — ← Another cysteine residue

49. E M B - R C G

50. E M B - R C G Non-covalent bonds

51. E M B - R C G Hydrogen bonds

52. The nitrogen atom involved in sharing belongs to one peptide bond, and the oxygen atom belongs to another peptide bond R R H R R | | | | | —CH —C—N—CH— —CH —C — N—CH — || || O O H | . . . . . . .

53. E M B - R C G Electrostatic bonds

54. E M B - R C G Hydrophobic bonds

55. E M B - R C G The structure of proteins can be considered to have four levels of organization : Primary structure Secondary structure Tertiary structure Quaternary structure

56. E M B - R C G

57. E M B - R C G Primary structure

58. Arg -Val- Cys -Ala-Tyr-Lys- Gly - Phe-Ser Arg -Val- Cys -Ala-Lys- Tyr - Gly - Phe-Ser Two different primary structures

59. E M B - R C G

60. E M B - R C G

61. E M B - R C G

62. This is the next higher level of organization The polypeptide chain i s twisted, turned and coiled to form various types of secondary structure Secondary structures include a -helix, b -pleated sheet, b -bend etc Secondary structure

63. E M B - R C G The polypeptide chain is coiled to form a helical structure The a -helix is produced by formation of hydrogen bonds between peptide linkages a -Helix

64. E M B - R C G

65. E M B - R C G

66. C H – C – N | R 7 | | O H | C | | O H | N R 6 — CH H | N H | N C || O CH— R 5 C || O H | N C H | R 3 H | N R 2 | CH C || O H | N C || O R 1 — CH R 4 — CH NH 2 C | | O Hydrogen bond

67. E M B - R C G

68. Portions of same peptide chain or different peptide chains running side by side are joined They are joined by hydrogen bonds formed between peptide linkages This produces an extended zigzag structure resembling a series of pleats b -Pleated sheets

69. The polypeptide chains forming the b -pleated sheets may be running: In opposite directions (N→C) forming anti-parallel b -pleated sheets or In the same direction forming parallel b -pleated sheets

70. Anti-parallel b -sheet

71. Parallel b -sheet

72. b -Bend

73. R n+2 ‒ C ‒ H R n+1 ‒ C ‒ H R n+3 N ‒ H O = C O װ C O װ C C װ O N ׀ H N ׀ H H ׀ N ׀ C ׀ H H ׀ C ׀ R n b -Bend EMB-RCG

74. E M B - R C G

75. E M B - R C G

76. a -Helix (ribbon ) b -Pleated sheets (arrows )

77. Tertiary structure

78. E M B - R C G

79. E M B - R C G EMB-RCG

80. The spatial arrangement of amino acid residues forming a specific three-dimensional conformation constitutes the tertiary structure of the protein Tertiary structure

81. Quaternary structure

82. E M B - R C G Joining of sub-units produces the quaternary structure of the protein Haemoglobin Examples of proteins having quaternary structure are: Creatinine kinase Lactate dehydrogenase

83. Quaternary structure Sub-unit Sub-unit

84. Quaternary structure Tertiary structure Secondary structure Primary structure

85. E M B - R C G EMB-RCG This leads to their denaturation or coagulation Protein structure may be disrupted by physical or chemical agents Disruption of structure causes loss of function Disruption of protein structure

86. Denaturation May be brought about by physical agents e.g. heat, x-rays, UV light etc or chemical agents e.g. acids, alkalis, heavy metals etc Secondary, tertiary and quaternary structures are disrupted Primary structure remains unaffected EMB-RCG

87. Heat Denatured protein Native protein

88. E M B - R C G EMB-RCG

89. E M B - R C G EMB-RCG Sometimes, it is possible to restore the denatured protein to its original structure and function This process is known as renaturation This is done by reversing the conditions that led to its denaturation

90. Native active ribonuclease Denatured inactive ribonuclease Renatured active ribonuclease Removal of urea and mercaptoethanol I I I I I I ‒ ‒ I I I I I I ‒ ‒ H 2 N H 2 N H 2 N COOH COOH HOOC Addition of urea and mercaptoethanol

91. Coagul ation When albumins and globulins are heated at their isoelectric pH, they are first denatured The subunits are separated and unfolded Unfolded polypeptides are then matted together to form a dense mass known as coagulum EMB-RCG

92. Coagulation is always irreversible Coagulated proteins are hydrolysed more easily than the native proteins These are coagulated on cooking, and become more digestible Milk and egg contain albumin and globulin

93. E M B - R C G If the primary structure is correct, the nascent protein will fold spontaneously It will automatically attain higher orders of structure and the correct conformation However, spontaneous folding is a slow process Rapid and correct folding of the protein is ensured by some enzymes and proteins Protein folding

94. E M B - R C G Enzymes involved in protein folding EMB-RCG

95. E M B - R C G Proteins involved in folding EMB-RCG

96. E M B - R C G EMB-RCG

97. E M B - R C G These enzymes and chaperones are also required to refold the proteins after they have passed through a membrane in the unfolded form EMB-RCG

98. E M B - R C G Misfolding of proteins EMB-RCG

99. E M B - R C G Misfolded proteins: May be non- functional May not reach their destination May be toxic EMB-RCG

100. E M B - R C G EMB-RCG

101. E M B - R C G EMB-RCG

102. E M B - R C G Alzheimer’s disease

103. E M B - R C G Creutzfeldt - Jakob Disease (CJD)

104. E M B - R C G EMB-RCG

105. E M B - R C G Acquired CJD

106. E M B - R C G They had misfolded prion protein in their brains Human beings who consumed beef from cows having mad cow disease developed a variant of CJD

107. E M B - R C G Fractionation of proteins EMB-RCG Separation of individual proteins from this complex mixture is required for academic, diagnostic or therapeutic purposes Biological materials contain a large number of proteins in addition to many non-protein components

108. E M B - R C G Several techniques of fractionation have to be employed in succession to obtain individual proteins in a pure form EMB-RCG Fractionation of proteins is a tedious and time consuming process

109. E M B - R C G EMB-RCG Methods for fractionation of proteins include: Salt fractionation Alcohol fractionation Centrifugation Electrophoresis Chromatography

110. E M B - R C G Salt fractionation For example, when a mixture of albumin and globulins is half-saturated with ammonium sulphate , globulins are salted out This process is known as salting out, and can be used for fractionation of proteins On treating a mixture of proteins with varying concentrations of salts, different proteins are precipitated at different salt concentrations

111. E M B - R C G The reverse process is known as salting in EMB-RCG On treating a mixture of two proteins with a salt concentration at which one protein is soluble and the other is not, the soluble protein will be dissolved or salted in

112. E M B - R C G Alcohol fractionation EMB-RCG Acetone can also be used for this purpose Thus, differential alcohol precipitation can be used for protein fractionation Different proteins are precipitated at different concentrations of alcohol

113. E M B - R C G Centrifugation EMB-RCG If a solution containing proteins is centrifuged at a high speed, the proteins are separated into different layers T he positions of different proteins depend upon their relative density

114. E M B - R C G EMB-RCG High-speed centrifugation is also known as ultra-centrifugation Ultra-centrifugation is frequently used for the separation of plasma lipoproteins

115. Chylomicrons – rin VLDL – rin I DL – rin LDL – rin H DL – rin

116. E M B - R C G Electrophoresis EMB-RCG They will be separated into different bands after sometime If particles differ in the number and type of charges, they will move at different rates in an electric field It is based on the movement of charged particles in an electric field

117. E M B - R C G In an electric field , different proteins migrate at different speeds and will form different bands after sometime EMB-RCG The bands can be visualized by staining them with suitable staining agents The stained bands can be quantitated by densitometry

118. Bands of serum proteins

119. Albumin Globulins b 1 b 2 a 1 a 2 g Densitometry of serum proteins

120. E M B - R C G Several types of electrophoresis have been developed EMB-RCG The supporting medium can be horizontal or vertical The support media, on which the sample is applied, may be paper, cellulose acetate, agar gel, starch gel, polyacrylamide gel etc

121. E M B - R C G EMB-RCG Different buffers are used to maintain pH as ionization of proteins is affected by pH In this technique, a high voltage (2,000 to 5,000 volts) is applied for a short period High voltage electrophoresis can be used for the separation of amino acids

122. E M B - R C G Chromatography EMB-RCG Basically, a chromatographic system consists of a stationary phase and a mobile phase Chromatography can be used for separation of proteins and several other compounds

123. E M B - R C G The mobile phase (liquid or gas) moves over the stationary phase (solid or liquid ) EMB-RCG When a mixture of substances is subjected to chromatography, the components are distributed between the two phases The distribution depends upon their relative affinities towards the two phases

124. E M B - R C G EMB-RCG The distribution generally depends upon two factors: Solubility Adsorptive affinity Accordingly , chromatography can be broadly divided into two types : Partition chromatography Adsorption chromatography

125. E M B - R C G In adsorption chromatography, the stationary phase is an adsorbent e.g. charcoal, alumina , silica gel etc EMB-RCG When the adsorbent is applied over a plate in a thin layer, it is known as thin-layer adsorption chromatography This can be spread over a glass or plastic plate or filled into a column

126. E M B - R C G The sample is then applied on the plate which is kept vertically in a glass tank EMB-RCG The mobile phase (liquid) is allowed to flow over the plate It can move upward (ascending chromatography) or downward (descending chromatography)

127. E M B - R C G EMB-RCG After sometime, they will be separated into different spots on the plate These can be stained and visualized The rate of movement depends upon their relative affinities towards the adsorbent D ifferent components of the mixture move with the mobile phase at different rates

128. E M B - R C G In partition chromatography, the stationary phase is a liquid supported on a solid medium The mobile phase is a solvent, generally non-polar A film of water molecules forms on paper and acts as the stationary phase A common form is paper chromatography in which the support medium is paper

129. E M B - R C G EMB-RCG These can be visualized by drying the paper and spraying it with a suitable staining agent After sometime, the components are separated forming distinct spots The rate depends upon their relative solubility in the mobile phase and the stationary phase Different components of the mixture migrate with the mobile phase at different rates

130. E M B - R C G The ratio of the distance travelled by a component to that travelled by the solvent is known as the Rf value of the component Rf = Distance travelled by the solute Distance travelled by the solvent EMB-RCG Rf values are useful in the identification of the compounds

131. E M B - R C G When the adsorbent is packed into a column , it is known as column chromatography EMB-RCG The mobile phase is then allowed to flow through the column The sample is layered over the column

132. E M B - R C G EMB-RCG These can be collected in different containers for identification and quantitation The time of emergence depends upon their relative affinities for the adsorbent D ifferent components of the mixture emerge from the column at different times

133. E M B - R C G Functions of proteins EMB-RCG Each protein has a unique conformation suited to its biological function Human beings synthesize thousands of different proteins As mentioned earlier, proteins perform a wide variety of functions

134. E M B - R C G EMB-RCG Function of a protein depends upon its structure A small change in primary structure can alter conformation and function of protein Thousands of inherited diseases occur due to synthesis of abnormal proteins Many of these are fatal and many others lead to severe clinical abnormalities

135. E M B - R C G Apart from some general functions, most proteins perform some specific functions EMB-RCG Depending upon their functions, proteins can be divided into a number of functional groups

136. Structural proteins

137. E M B - R C G

138. E M B - R C G

139. Each polypeptide chain is coiled into a left-handed helix in which three amino acid residues are present in each turn

140. E M B - R C G

141. E M B - R C G Coiling of three left-handed helices into a right-handed triple helix increases the tensile strength Tensile strength is further increased by cross-links between: The three chains Triple helices running parallel

142. E M B - R C G R CH 2 ‒NH 2 R CHO →

143. Two aldehyde groups may undergo aldol condensation resulting in cross-linking CH ‒ CH 2 II O CH ‒ CH 2 II O Polypeptide CH 2 ‒ CH II O CH 2 ‒ CH II O CH ‒ CH II O CH ‒ CH II O CH 2 CH 2 CHOH CHOH Polypeptide Polypeptide Polypeptide

144. Schiff bases are formed between : Aldehyde groups of modified lysine and hydroxylysine residues e -Amino groups of unmodified lysine and hydroxylysine residues Cross-linking may also occur due to formation of Schiff bases

145. E M B - R C G

146. E M B - R C G

147. E M B - R C G

148. E M B - R C G

149. E M B - R C G Oxidative deamination of lysine and hydroxylysine residues also occurs after translation Oxidative deamination is catalysed by lysyl oxidase, a copper-containing enzyme This is followed by cross-linking by aldol condensation or formation of Schiff bases Cross-linking converts tropocollagen into collagen

150. E M B - R C G Abnormal collagens may be formed due to mutations or nutritional deficiencies Genes encoding enzymes involved in post-translational modifications Genes encoding collagens or Genetic defects leading to the formation of abnormal collagens may involve:

151. E M B - R C G Different types of Ehlers- Danlos syndrome result from abnormal collagens in which: Joints are hyper-mobile Skin is hyper-elastic Tissues are fragile

152. E M B - R C G

153. E M B - R C G

154. E M B - R C G Defective cross-linking may also occur due to binding of: Homogentisic acid to collagen in alkaptonuria Homocysteine to collagen in homocysteinuria

155. E M B - R C G Catalytic proteins

156. E M B - R C G

157. E M B - R C G Membrane transport proteins

158. E M B - R C G Membrane channels

159. E M B - R C G Contractile proteins

160. E M B - R C G Receptors

161. E M B - R C G Signal transducers

162. E M B - R C G Storage proteins

163. E M B - R C G Carrier proteins

164. E M B - R C G EMB-RCG

165. E M B - R C G Antibodies

166. E M B - R C G

167. E M B - R C G Complement proteins

168. E M B - R C G Coagulation factors

169. E M B - R C G Lubricant proteins

170. Thank you