PROTEIN STRUCTURE & FUNCTION by Dr DKV 2023.ppt
- 1. Dr. DKV Prasad Associate Professor
- 2. Composition of proteins • Proteins are macromolecules composed of many amino acids. • The sequence of the amino acids determine each proteins unique 3 dimensional structure and its specific function. • Constitute ¾ th of dry weight of body • Used for building of body and abnormalities in protein structure leads to molecular diseases leading to altered metabolic functions • Proteins contain nitrogen in addition to C,H,O which are present in carbohydrates and lipids. • The nitrogen content is 16% of the molecular weight of proteins. • Small amounts of Sulphur and phosphorous are also present.
- 3. Classification of proteins • Based on – functions – composition and solubility – shape – nutritional value
- 4. • Catalytic proteins – enzymes, pepsin,lipase • Structural proteins – collagen, elastin • Contractile proteins – myosin, actin • Transport protein – hemoglobin, myoglobin, albumin, transferrin • Regulatory protein/hormones – ACTH, insulin, growth hormone • Genetic proteins – Histones • Protective proteins – Immunoglobulins, interferons, clotting factors Classification of proteins based on function
- 5. Classification of proteins • Based on – functions – composition and solubility – shape – nutritional value
- 6. • Simple proteins • Conjugated proteins • Derived proteins Classification of proteins based on Composition & Solubility
- 7. Classification of proteins based on Composition & Solubility Proteins Simple proteins Conjugated proteins Derived proteins •Albumins •Globulins •Protamines •Prolamines: Gliadin •Lectins •Scleroproteins: Hair,horns •Glycoproteins: Blood group antigens •Lipoproteins: •Nucleoproteins: Histones •Chromoproteins: Visual purple •Phosphoproteins:Casein •Metalloproteins: Ferritin •Peptones •Peptides
- 8. They are degradation products of native proteins Primary derived: denatured/coagulated proteins. E.g, proteans, metaproteins Secondary derived proteins: progressive hydrolytic products of proteins cleaving peptide linkage E.g; proteoses, peptones, peptides Derived Proteins :
- 9. Classification of proteins • Based on – functions – composition and solubility – shape – nutritional value
- 10. 1. Globular proteins – albumin , globulin 2. Fibrous proteins – collagen, elastin Classification of proteins based on Shape :
- 11. Classification of proteins • Based on – functions – composition and solubility – shape – nutritional value
- 12. 1. Nutritionally rich proteins – Complete proteins (casein) 2. Incomplete proteins – lacks one essential amino acids (pulses deficient in methionine) 3. Poor proteins – Lacks many essential amino acids Classification of proteins based on Nutritional value :
- 13. Structural organization of Protein Conformation of protein • The folding of the polypeptide chain of a protein into specific three dimensional structure is referred to as the conformation of protein • Exact conformation is determined by its amino acid sequence • Specific biological function depends upon conformation
- 14. Different levels of protein structure • PRIMARY • SECONDARY • TERTIARY • QUATERNARY
- 15. PRIMARY STRUCTURE • Sequence / order of aminoacids in protein (gly-ala-val-met) • If primary structure is known - total number of amino acid residues - N and C terminals - sequence of amino acids & their types • The defined arrangement of amino acids gives the protein its properties and determines biological activity. • Substitution of an amino acid can reduce or abolish biologic activity (eg: sickle cell hemoglobin) • Bonds responsible – peptide, disulphide (covalent, permanent)
- 16. PEPTIDE BOND and Peptides • Peptides are oligomers of amino acids formed by the condensation of an amino group of one amino acid with the carboxyl group of another amino acid. • Dipeptide – Two amino acids • Tripeptide – Three amino acids H2N – C- COOH + H2N-C – COOH H2N – C- CO – NH- C – COOH H R 1 H H H R 2 R 1 R 2 Peptide bond Amino end N- terminal Carboxyl end C-terminal Numbering and naming NH2-Gly-Ala-COOH
- 17. PEPTIDE BOND
- 18. • Tetrapeptide – four aminoacids • Oligopeptide – upto 10 aminoacids • Polypeptide -10 to 50 aminoacids • > 50 aminoacids – protein • 20 aminoacids form 20ⁿ possibilities of combination of aminoacids in protein (n= no of aminoacids in protein)
- 19. • The peptide bond has partial double bond and partial single bond character – no rotation • 1.32 A • Trans configuration – stearic hindrance of R group • Alternated between C α -N and C-Cα • Rotation is possible around C α -N and C-Cα but not between C-N bond • Partial double bond – limited polypeptide chain folding • Side chains rotate freely at angles of rotation- Ramachandran angle. Characteristics of Peptide bond
- 20. Primary Structure of Protein • Disulphide bonds / bridges formed between 2 cysteine residues – form branching points • S-S bonds between different polypeptide chains in same protein – inter chain • S-S bonds between same polypeptide chain – intra chain • Pseudopeptide – instead of alpha COOH group , gamma carboxyl group enters into peptide bond (Glutathione)
- 21. Importance of primary structure • It permits comparison between normal and mutant protein. • It helps in positioning the different organism on the evolutionary tree by comparing the same protein in different species. • To know the three dimensional structure of the protein • It permits the synthesis of peptides and proteins in large amounts by chemical synthesis and recombinant DNA technology to aid therapy
- 22. Primary structure of Insulin
- 23. Secondary structure of Protein • Folding of polypeptide chains due to non covalent bonding between neighbouring / closely placed aminoacids (3-4aa apart) in primary structure • Leads to folding patterns in protein chain which occur either regularly / occasionally • Regular patterns – 2 patterns • Secondary structure and higher levels are stabilized by large number of noncovalent forces (hydrogen bonds, electrostatic bond, hydrophobic interactions, vander waal’s forces)
- 24. • Hydrogen bond – weak electrostatic attraction between electronegative atom O/ N and H atom (donated by –OH, NH2, COOH) • Electrostatic bonds (ionic bond) – positive charges provided by arginine, lysine and negative charges provided by aspartic acid and glutamic acid) •Hydrophobic bonds – interactions between nonpolar hydrophobic side chains by eliminating water molecules •Vanderwaals forces – weak but maximum stability of protein structure
- 25. Secondary structure of Protein VARIOUS TYPES OF SECONDARY STRUCTURE • helix • -pleated sheet Super secondary structure • unit • corner • twisted sheets • barrel
- 26. Secondary structure – α Helix • The polypeptide is in helical or coiled/spiral form • Most common and stable conformation of protein • Long fibrous proteins – collagen, keratin • Hemoglobin, myoglobin – abundant • Polypeptide with α-helix has - • Can be right handed or left handed helix (right handed helix commonest) • folded helically around central axis • one turn - 0.54 nm & has 3.6 AA residues
- 27. - helix stabilized by hydrogen bond between N atom of one peptide bond and carboxyl O atom of 4th peptide bond (1st and 4th , 2nd and 5th ) - Polypeptide bonds forms backbone & side chains extends outwards - proline and hydroxy proline - α-helix breakers • Stability is attained by extensive cross linking of chains by disulphide bonds.
- 28. • β pleated sheets formed when polypeptide chain / parts align longitudinally • It is an fully extended structure • Can form between different regions of same polypeptide chain/ between different polypeptide chains • Polypeptide chains/regions are placed parallel • Hydrogen bonds formed between peptide bonds similar to alpha helix with relatively apart aminoacid sequence • The amide N and Carbonyl O are extended at nearly right angles to the long axis of polypeptide chain. • β pleated sheets can exist as parallel / antiparallel sheet Secondary structure – β - pleated sheet
- 30. • Valine, isoleucine, and phenylalanine enhance -Sheets • Proline doesn’t fit well into -Sheets • Different regions of same polypeptide can have both alpha helix and beta sheets • Other secondary sructures - loops/coils - turns or bends Secondary structure – β - pleated sheet
- 31. super secondary structure α units - meander unit Greek key barrel
- 33. Tertiary structure of Protein • Over all three dimensional arrangement of whole polypeptide chain • AA residues placed far apart in primary structures or those present in different secondary structure domains interact – tertiary • combination of various secondary structures and non ordered segments into ordered form. • Its three dimensional conformation brings most of the nonpolar amino acids to the interior where they have minimal contact with water. The polar side chains tend to move to the surface of protein (in case of hydrophilic protein)
- 34. Tertiary structure (bonds involved) • Van der Waal’s forces, hydrogen bonding, salt bridges, hydrophobic bonds, electrostatic bonds and disulphide bonds • Tertiary structure- lowest energy and highest stability state of polypeptide chain • The accurate three dimensional structure is produced by correct folding of the protein assisted by Chaperones. • Incorrect folding may produce altered proteins, which is the basis of Prion diseases
- 36. Quarternary structure • Proteins having more than one polypeptide chain (2/ more) • Each polypeptide chain is called subunit/ monomer. • Quaternary - It is the spatial arrangement of the various subunits of a protein molecule. • Monomer units can be – homopolymer/hetero • The forces responsible for the formation and maintenance of quarternary structure are the same as those that create secondary and tertiary structures. • Hemoglobin – best example – having 2 alpha 2 beta chains • Myoglobin – one chain – only primary, secondary, tertiary structures
- 39. Protein folding • Unique structure of protein involves – folding of polypeptide chains • Information of folding is present in the primary structure- sequence of AA • Folding occurs spontaneously but not all proteins • Few require accessory proteins –chaperones to promote folding • Folding is hierarchial manner - focal small parts of chain undergo structural folding to alpha and beta structures - interactions occur between secondary structures to domains - conformational change in multi domain protein to mature monomeric units - monomeric units interact to form quaternary protein
- 40. • Assisted folding by chaperones and certain isomerases • Chaperones : - proteins which interact with partially folded/improperly folded nascent proteins to facilitate correct folding - 2 classes – Hsp70, chaperonins a) Hsp70 (heat shock proteins) as they are abundant in cells stressed by heat - they protect proteins from denatured - they bind to specific hydrophobic regions of nascent polypeptide and shield them from environment and prevent improper folding and aggregation of proteins - requires ATP b) Chaperonins (Hsp 60) - similar but present in micro organisms
- 41. Prion diseases • neurodegenerative diseases caused by misfolded proteins • Dementia, loss of coordination, encephalitis • Also called spongiform encephalitis • Best example is Mad cow disease/ bovine spongiform encephalitis and Creutzfeldt disease in humans • Causative agent is prion protein which on mutation causes change in alpha helix to beta sheets
- 42. Protein denaturation • Defined as unfolding and disorganization of protein structure by disruption of hydrogen bonds, hydrophobic bonds and electrostatic bonds • Disruption of secondary, tertiary and quaternary structures • Primary structure is not affected • Effects of denaturation – Loss of biological activity, least soluble – Denatured protein are easily cooked – Denatured protein are sometimes renatured when the physical agent is removed. eg ribonuclease
- 43. Protein denaturation • Agents which cause denaturation - Heat, strong acids/ bases - Urea / guanidine hydrochloride, Salicylate - X-rays, UV rays - Detergents, Ions of heavy metals
- 44. Heat Coagulation • Protein denaturation caused by heat at high temperatures is irreversible • A thick floating conglomerates is formed called coagulum • Proteins like albumin and to some extent globulin gets denatured when heated at iso-electric point. • Denatured protein may be soluble and can be made to precipitate by adding acetic acid. • This reaction is used to identify the excretion of albumin in urine
- 45. Steps for determining the primary structure • Determination of the number of polypeptide chains in a protein • Separation and purification of individual polypeptide chains • Determination of amino acid composition by complete hydrolysis of the polypeptide chains • Identification of N-terminal and C-terminal amino acids. • Site specific hydrolysis of polypeptide chain using specific enzymes to get a mixture of overlapping peptides • Separation and purification of each of these peptides • Ascertaining the position of disulphide bridges if any.
- 46. Determination of Secondary and Tertiary Structures • X-ray crystallography studies • Optical rotatory dispersion Determination of quaternary structure • Ultracentrifugation • Sucrose density gradient centrifugation • Polyacrylamide gel electrophoresis
- 47. Separation technique CHROMATOGRAPHY The term is derived from the Greek word “Chroma” meaning colour. This method was first employed by Tswett in 1903. Paper chromatography & Thin layer chromatography Both these are Liquid – Liquid partition chromatography. Paper Chromatography is widely used to separate amino acids and sugars. Thin layer chromatography is used to separate lipids.
- 48. Separation techniques Paper chromatography Types – Ascending & Descending Stationary phase – water held on a solid support. Mobile phase – Butanol –acetic acid –water Principle of separation The difference in distribution of the amino acids / sugars between two phases. Visualisation of chromatography – after the chromatographic run the paper is dried and it is sprayed with ninhydrin for amino acids, sulphuric acid for phospholipids and diphenylamine for sugars Importance of Rf - It helps in identification of the unknown Rf = Distance traveled by solute ------------------------------------ Distance traveled by solvent
- 51. Biologically important peptides Name Amino acid residues Source & Function Thyrotropin releasing hormone 3 Secreted by hypothalamaus, causes anterior pituitary to release thyrotropic hormone Vasopressin 9 Secreted by posterior pituitary, causes kidney to retain water from urine Enkephalin 5 Opiate-like peptide found in brain that inhibits sense of pain. Angiotensin I, II 10 8 Hypertensive peptide – stimulates the release of aldosterone from adrenal cortex Glutathione 3 Metabolism of xenobiotics
- 52. Name Amino acid residues Source and function Bradykinin 9 Vasodilator Substance P 10 Neurotransmitter Tyrocidin, gramicidin Cyclic peptides (10) Polypeptides antibiotics Aspartame 2 Sweetening agent in many beveragers Biologically important peptides
- 53. Study Texts • Textbook of Biochemistry – DM Vasudevan • Illustrated Biochemistry – Lippincott • Harper’s biochemistry References
- 54. Thank you