Structural level of organization of proteins

description of different levels of structural organization of proteins and renaturation and denaturation of proteins

1. levels of protein Structure organization D.Indraja

2. The structure of protein is organized in to 4 levels of organization they are: Primary structure Secondary structure Tertiary structure Quaternary structure These levels also reflect their temporal sequence. Proteins are synthesized as a primary sequence and then fold into secondary → tertiary → and quaternary structures.

3. Primary structure of proteins It was given by sanger Primary structure of protein refers to the  linear sequence and arrangement of amino acids in polypeptide chain. Amino acids are building blocks of proteins. The amino acids are linked with each other with the help of Peptide bond to form a peptide (or) Protein Thus bond is formed between -NH2 group of one amino acid and -COOH group of the next amino acid. During peptide bond formation, one water molecule will be eliminated, hence it is called as dehydration synthesis. The end at which –COOH group is free is called C-terminal and other end at which –NH2 group is free is called N-terminal of polypeptide chain Due to partial double bond character between –CO and –NH group, they do not rotate

4. The primary structure of protein is stabilized by peptide bonds and disulphide bonds. These are strong covalent bonds. The disulphide bond is formed between cysteine residues. Eg: insulin insulin Contain 51 Amino aids which are arranged in 2 peptide chains A chain - 21 Amino acids B chain - 30 Amino acids These two chains an linked by disulphide bonds.

5. Secondary structure of Proteins It was given by Linus Pauling and Robert Corey in 1951 by using X-ray diffraction studies Amino acids that are located near to each other interacts to form regular arrangement called secondary structure. The secondary structure of protein is more compact and stable than primary structure The secondary structure of proteins is stabilized by hydrogen bonds The hydrogen bond is between the “o” atom of c=o group of one amino acid and H atom of N-H group another amino acid polypeptide chain There are three commonly occurring secondary structure. They are; α-Helix β-sheet β-bends or β-turn

6. α-helix structure: α-helix is a right handed helical structure formed by twisting of polypeptide chain. It is a spiral structure. Each helix in α-helix structure contains 3.6 aminoacids residues. Vertical length of each helix is known as pitch which is 5.4 Å. Therefore, the vertical distance between two nearest aminoacids is 1.5Å. In α-helix, -C=O group of each aminoacid is hydrogen bonded with –NH group of other aminoacid which is situated four amino acid ahead. Which is (i+4) or (n+4) arrangement Therefore, -C=O and –NH group of all aminoacids are hydrogen bonded in α-helical structure. R-group of aminoacid in α-helix are projected outward to minimize steric hindrance Some aminoacids disrupt α- helix. For examples, the aminoacids with charged R-group disrupt α- helix by electrostatic repulsion or by formation of ionic bond. Similarly aminoacids with bulky R-group disrupt α- helix by steric interference. Aminoacids glycine and proline bring bend in polypeptide chain and disrupt α- helix

7. Secondary structure of proteins- α helix

8. β-sheet structure: β-Sheet is the most stable form of secondary structure of protein. It is formed between two different polypeptide chains which are placed parallel or antiparallel to each other. It can also be formed by folding of same polypeptide chain. In β-sheet structure, two polypeptide backbone are linked with each other by H-bond which are formed between –CO and –NH group. R-group of amino acids are alternately projected above and below the plane of β-sheet. The surface of β-sheet is not straight but it is pleated. Therefore, it is also known as β-pleated sheet. Polypeptide backbone in β-sheet is extended rather than being tightly coiled as in α-helix. The axial distance between two nearest amino acid is 3.5 Å in contract with 1.5 Å in α-helix. In β-sheet hydrogen bond may be inter chain or intra chain but they are always inter chain in α-helix.

9. Secondary structure of proteins- β pleats

10. β-bends or β-turn structure: β-bend reverse the direction of polypeptide chain and helps it to form globular (spherical ) structure. Β-bend structure consists of at least 4 amino acids in which nth amino acid is hydrogen bonded with (n+3) th amino acids. Glycine and proline are always found in β-bend structure. Ring of Proline attached with α-carbon atom helps to bend the chain. Similarly, lack of R-group in glycine permits great degree of rotation around α-carbon atom and bring bend in the polypeptide chain.

11. Tertiary structure of protein: It was given by John Kendrew and his colleagues in 1980 by using X-ray analysis It is more compact and folded structure than Secondary structure Tertiary structure refers to the overall folding of a polypeptide chain to form a final three dimensional structure. For example, a globular protein which are larger than 200 amino acids units forms two or more domains by folding of polypeptide chain by either α-helix, or β-pleated sheet or β-bend. Finally these domains associates with each other to form final 3D structure. Therefore, tertiary structure refers to the formation of these domains by overall folding of polypeptide chain and then final association of these domains to form globular 3D structure

12. This protein structure is mostly stabilized by non covalent interactions they are Hydrophobic interactions Hydrophilic interactions Vanderwal interactions Hydrogen bonds Disulphide bonds Hydrophobic interactions These are weak bonds. These are formed in a polypeptide chain in between the nonpolar R group containing aminoacids Hydrophilic interactions These are weak bonds. These are formed in a polypeptide chain in between the polar R group containing aminoacids

13. Vanderwal interactions These are weak bonds. These are formed in a polypeptide chain in between the hydrophobic aminoacids Hydrogen bonds These are weak bonds. These are formed in a polypeptide chain in between the NH2 & C=O groups Disulphide bonds These are strong covalent bonds. These are formed in a polypeptide chain in between the cysteine-cysteine aminoacids

14. Quaternary structure of protein: It was given by max Perutz and john kendrew in 1959 Some proteins are composed of more than one polypeptide chain. Each polypeptide chain in such protein are called sub-units. The quaternary structure refers to interaction between these sub-units to form large final 3D structure. Therefore, quaternary structure is interaction between different polypeptide chains of multi chain protein. Quatarnary structure is found only in protein which are composed of more than one polypeptide chains such as hemoglobin Bonds like H-bond, ionic bond, hydrophobic interaction helps to from quaternary structure. Examples of quaternary structure of protein are hemoglobin , DNA polymerases and ion channels .

15. Denaturation The native proteins are said to be the proteins occurring in animal and plant tissues. They pos­sess many characteristic properties such as solubil­ity, viscosity, optical rotation, sedimentation rate, electrophoretic mobility etc. For an oligomeric protein, denaturation may involve dissociation of the protomers with or with­out subsequent unfolding or with or without un­dergoing changes in protomer conformation. Denaturation of Proteins: Denaturation may be defined as the disruption of the secondary, tertiary and quarternary structure of the native protein resulting in the alterations of the physical, chemical and biological characteristics of the protein by a variety of agents.

16. Denaturing Agents: 1. Physical agents: Heat, surface action, ul­traviolet light, ultrasound, high pressure etc 2. Chemical agents: Acids, alkalis, heavy metal salts, urea, ethanol, guanidine de­tergents etc. Urea and guanidine probably interfere with the hydrogen bonds between peptide linkages in the secondary and tertiary struc­ture of proteins Physical Alterations: Many proteins, especially of the globular type, can be crystallized in the native state. But dena­tured proteins cannot be crystallized Chemical Alterations: The denatured protein is greatly decreased in solubility at its isoelectric point due to disruption of native configuration

17. Acid and alkali  disrupts hydrogen bonds in protein Urea, detergents  disrupt hydrophobic bonds in protein β mercapto ethanol and performic acid  disrupts disulphide bonds between cysteine residues Biological Alterations: The digestibility of certain denatured proteins by proteolytic enzymes is increased. Enzymatic or hormonal activity is usually destroyed by dena­turation. The antigenic or antibody functions of proteins are frequently altered Irreversible denaturation

18. If the denaturation is severe, the protein mol­ecules become insoluble and precipitation results as well as the changes in the properties of the pro­teins are permanent and “irreversible”. In case of mild denaturation, there is “reversible denatura­tion” leading to the slight changes in the proper­ties of the protein which can be restored to the na­tive state after suitable treatment Significance: 1. The precipitation of the native protein as a result of denaturation is used to advan­tage in the clinical laboratory. 2. Blood or serum samples to be analysed for small molecules (e.g., glucose, uric acid, drugs) generally are first treated with ac­ids such as trichloroacetic acid, phosphotungstic acid or phosphomolybdic acid to precipitate most of the proteins present in the sample. This is removed by cen­trifugation and the protein-free supernatant liquid is then analysed .

19. Renaturation The process by which the denatured proteins regain their native confirmation is called renaturation The denaturation is reversible in some cases It is carried out by Christian Anfinsen in 1951 Example : Bovine ribonuclease contain 124 A.A . It is native configuration When the protein is treated with 8m urea and β - mercaptoethanol is denatured and produce 8 cysteine residues urea disrupt the hydrophobic interaction of Rnase where as β - mercaptoethanol disrupts the disulphide bond

20. When the Rnase is free from the urea β - mercaptoethanol by dialysis it is slowly regain its enzymatic activity and contain 4 disulphide bonds RNase RNase RNase Urea + β - mercaptoethanol filteration Urea + β - mercaptoethanol