3. protein structure


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3. protein structure

  1. 1. Protein 3-Dimensional Structure and Function
  2. 2. Terminology • Conformation – spatial arrangement of atoms in a protein • Native conformation – conformation of functional protein
  3. 3. Protein Classification • • • • One polypeptide chain - monomeric protein More than one - multimeric protein Homomultimer - one kind of chain Heteromultimer - two or more different chains (e.g. Hemoglobin is a heterotetramer. It has two alpha chains and two beta chains.)
  4. 4. Protein Classification(chemical nature and solubility) • Simple – composed only of amino acid residues • Conjugated – contain prosthetic groups (metal ions, co-factors, lipids, carbohydrates) Example: Hemoglobin – Heme
  5. 5. Protein Classification – simple proteins Fibrous – 1) 2) 3) 4) polypeptides arranged in long strands or sheets water insoluble (lots of hydrophobic AA’s) strong but flexible Structural (keratin, collagen, elastins) Globular – 1) 2) 3) 4) polypeptide chains folded into spherical or globular form water soluble contain several types of secondary structure diverse functions (enzymes, regulatory proteins)
  6. 6. catalase keratin collagen
  7. 7. Protein Function(classification) • • • • • • • • • Catalysis – enzymes(hexokinase, pepsin) Structural – keratin Transport – hemoglobin Trans-membrane transport – Na+/K+ ATPases Toxins – rattle snake venom Contractile function – actin, myosin Hormones – insulin Storage Proteins – seeds and eggs Defensive proteins – antibodies
  8. 8. Protein classification(nutrition) • 1. Complete protein –egg albumin, milk • 2. Partially incomplete protein (limiting lys. & thr) – wheat and rice protein • 3. Incomplete protein – gelatin(lacks Trp)
  10. 10. ORGANISATION OF PROTEINS a) Primary Structure of a protein is the linear sequence of amino acids & location of disulphide bonds of its polypeptide chain or chains (if it comprises more than one polypeptide). Only covalent peptide bond b) Secondary Structure is the local spatial relationship of neighboring amino acid in the polypeptide backbone atoms without regard to the conformations of its side chains. c) Tertiary Structure- the 3 D structure of an entire polypeptide. Describes the folding of the secondary structural elements and specifies the position of each atom including those of the side chains. d) Quaternary Structure refers to the spatial arrangement of the polypeptides (subunits) if present in a
  11. 11. 4 Levels of Protein Structure
  12. 12. Organization of Proteins a) Primary Structure (10) of a protein refers to the linear number and order of the amino acids present and location of disulphide bonds of its polypeptide chain or chains (if it comprises more than one polypeptide) Primary structure determines Biological activity Designation= N-terminal end on the left as No 1 with free αamino gp), C-terminal end a free a-carboxyl group) is to the right. Sequence predetermined by the nucleotide sequence of the gene. Covalent peptide bond is the only type of bond linking amino acids.
  13. 13. PEPTIDE BOND • The peptide bond has a partial double-bond character it is shorter than a single bond and is therefore RIGID and PLANAR [This prevents free rotation around the bond between the carbonyl carbon and the nitrogen of the peptide bond] • The peptide bond is generally a trans bond. [There will be steric interference of R-Group if it is in Cis position.] • The – C =O and –NH groups of the peptide bond are polar and are involved in hydrogen bonds but they are uncharged [they neither accept nor give off protons]
  14. 14. 1o Structure Determines 2o, 3o, 4o Structure • Sickle Cell Anemia – single amino acid change in hemoglobin related to disease • Osteoarthritis – single amino acid change in collagen protein causes joint damage
  15. 15.  2O Structure is the local spatial relationship of neighboring amino acids in a polypeptide.  An important characteristic of 2o structure is the formation of hydrogen bonds between –CO group of one peptide and the –NH group of another nearby peptide Eg. 1. α helix 2. β- pleated sheet 3. β bend
  16. 16. 2o Structure Related to Peptide Backbone •Double bond nature of peptide bond cause planar geometry •Free rotation at N - αC and αCcarbonyl C bonds •Angle about the C(alpha)-N bond is denoted phi (φ) •Angle about the C(alpha)-C bond is denoted psi (ψ) •The entire path of the peptide backbone is known if all phi and psi angles are specified
  17. 17. Ramachandran Plots •Describes acceptable φ/ψ angles for individual AA’s in a polypeptide chain. •Helps determine what types of 2o structure are present
  18. 18. Alpha-Helix • First proposed by Linus Pauling and Robert Corey in 1951 • Identified in keratin by Max Perutz • A ubiquitous component of proteins • Stabilized by H-bonds
  19. 19. Alpha-Helix •Residues per Right handed turn: 3.6 helix •Rise per residue: 1.5 Angstroms •Rise per turn (pitch): 3.6 x 1.5A = 5.4 Angstroms •amino hydrogen H-bonds with carbonyl oxygen located 4 AA’s away forms 13 atom loop
  20. 20. Alpha-Helix All H-bonds in the alpha-helix are oriented in the same direction giving the helix a dipole with the Nterminus being positive and the C-terminus being negative
  21. 21. Alpha-Helix •Side chain groups point outwards from the helix •AA’s with bulky side chains less common in alpha-helix •Glycine and proline destabilizes alphahelix
  22. 22. Amphipathic Alpha-Helices + One side of the helix (dark) has mostly hydrophobic AA’s Two amphipathic helices can associate through hydrophobic interactions
  23. 23. Beta-Strands and Beta-Sheets • Also first postulated by Pauling and Corey, 1951 • Strands may be parallel or antiparallel • Rise per residue: • – 3.47 Angstroms for antiparallel strands – 3.25 Angstroms for parallel strands – Each strand of a beta sheet may be pictured as a helix with two residues per turn
  24. 24. Beta-Sheets • Beta-sheets formed from multiple side-byside beta-strands. • Can be in parallel or anti-parallel configuration • Anti-parallel betasheets more stable
  25. 25. R R R R R R R R R R R R R R
  26. 26. Beta-Sheets • Side chains point alternately above and below the plane of the beta-sheet • 2- to 15 beta-strands/beta-sheet • Each strand made of ~ 6 amino acids
  27. 27. Loops and turns Loops • Loops usually contain hydrophillic residues. • Found on surfaces of proteins • Connect alpha-helices and beta-sheets Turns • Loops with < 5 AA’s are called turns • Beta-turns are common
  28. 28. Beta-turns • allows the peptide chain to reverse direction • carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away • proline and glycine are prevalent in beta turns
  29. 29. Supersecondary Structures (Motifs) • Certain combinations of secondary structures (α-helix & β-pleated sheet) can be observed in different folded protein structures called supersecondary structures. • They are also called structural motifs. Eg. helix-turn-helix • α-Helix and β-pleated sheet are combined in many ways as the polypeptide chain folds back on itself in a protein. • Glycine & proline are frequently encountered in reverse turns, at which polypeptide chain changes direction. • Eg. βαβ unit - 2 parallel strands of β-sheet is connected by a stretch of α-helix. β-sheet β-sheet
  30. 30. Common Motifs
  31. 31. Tetriary structure of a protein is the 3-D structure of a protein. The 3-D structure depends on; Folding of the secondary structural elements Geometric relationship between distant segments of primary structure. Relationship of the side chains with one another in three-dimensional space. • Major cohesive forces in tertiary structures are noncovalent forces such as hydrophobic interactions between amino acid side chains. • Other forces – electrostatic, H-bonding, S-S disulphide bridges • In water-soluble globular proteins, surface is occupied by hydrophilic side chains, while the nonpolar residues form the hydrophobic core.
  32. 32. Three-dimensional folding of the protein myoglobin
  33. 33. NONCOVALENT FORCES  Hydrogen bonds : (a) are formed by sharing of a hydrogen between 2 electron donors. (b) Hydrogen releasing groups are –NH [of imidazole, indole and peptide] -OH [serine and threonine] –NH2 [lysine and Arginine] (c) Hydrogen accepting groups are COO- [aspartic, Glutamic C=O [peptide and S-S [disulphide]  Electrostatic bonds: [ Ionic bonds]: (a) are the βattractive forces between 2 opposite charges or repulsion between 2 similar charges (b) Positive charges are produced by lysine, arginine and histidine (c) Negative charges are provided by βand ‫ ץ‬carboxyl groups of aspartic acid and glutamic acid
  34. 34. NONCOVALENT FORCES • Hydrophobic bonds: are formed by interactions between nonpolar hydrophobic side chains by eliminating water molecules • Van der Waals forces: (a) are attractive forces operating between all atoms due to oscillating dipoles. (b) Although very weak, vander waals forces collectively contribute maximum towards the stability of protein structure
  35. 35. d) Quaternary Structure ( 40 ) • Formed by interaction between different polypeptides (subunits). • Quaternary structure found in proteins with more than one polypeptide • Subunits held together by non-covalent interactions and/or inter chain disulphide bonds. Eg. Hemoglobin consists of 2 α and 2 β subunits
  36. 36. The 4 levels of Protein Structure
  37. 37. Protein Folding Pathways Random or Directed?  Evidence indicates that proteins fold to their native conformations via directed pathways rather than via random manner. Not well understood. Directed Pathway for Protein Folding • Protein folding begins with formation of local segments of secondary structure (α helices and β sheets). Very rapid process. • These segments condense to resemble the secondary structure of native protein. • Secondary structures become stabilized and 3o structures begin to form. • Protein undergoes complex motions to form the hydrophobic core • In multi-subunit protein, the subunits associate through slight conformational adjustments to form the quaternary structure..
  38. 38. Protein Folding Pathways cont……. • Folded protein usually are quite compact but bonds may be available to bind metal ions, cofactors, substrates etc which can increase stability of protein. • If sulphydryl groups are located on the surface or near the points of contact between the associating species, the formation S-S linkages can serve to stabilize the structures.  Protein disulphide isomerases catalyse the breakage and formation of disulphide bonds so that incorrect linkages are not stabilised and allowing stable ones to be formed. •. Chaperone proteins or Chaperonins bind to unfolded or partiallyfolded polypeptide chains. Prevents improper association of exposed segments which can lead to non-native folding, polypeptide aggregation or precipitation.
  39. 39. ERROR IN PROTEIN FOLDING AND DISEASES • Misfolded versions of proteins normally present in the same tissues can aggregate among themselves and form fibrous deposits. In the brain, this gives rise to a number of neurological diseases. • Alzheimer’s Disease Fibrous proteins or plaques are deposited in brain of patients and is due to alteration of a normal protein. The neurofibrillary tangles are paired helical filaments made up of Protein.
  40. 40. Error in Protein Folding and Diseases 2. Bovine spongiform encephalopathy (“mad cow disease”) ( Creutzfeldt-Jacob Disease) Proteins normally found in brain and nervous system [PrP] change into some abnormal protein [PrPsc] known as Prions. They have normal primary structure but abnormal tertiary structure. The abnormal proteins convert normal proteins into abnormal varieties, producing a chain reaction that generates new infectious materials. The normal protein PrP is 253 aminoacids, is soluble and can be digested by lysosomal enzymes but abnormal PrPsc is insoluble and cannot be digested and is accumulated inside nerve cells which damage brain cells
  41. 41. Denaturation • The phenomenon of disorganization of native protein structure is known as denaturation.Denaturation results in the loss of secondary,tertiary and quaternary structure of proteins .This involves a change in physical ,chemical and biological properties of protein molecules.
  42. 42. Agents of denaturation • Physical agents-Heat,voilent shaking,xrays,UV radiation. • Chemical agents-Acids,alkalies,organic solvents,urea,salicylate.
  43. 43. Characteristics of denaturation • • • • Native helical structure of protein is lost. Primary structure of a protein with peptide linkage remains intact Protein loses it’s biological activities. Denatured protein becomes insoluble in solvent in which it was originally soluble. • Viscosity of denatured protein increases while it’s surface tension decreases. • Denaturation is associated with increase in ionizable and sulfahydryl group of protein.this is due to loss of hydrogen and disulfide group.
  44. 44. Characteristics of denaturation • Denatured protein is more easily digested. this is due to increase exposure of peptide bonds to enzymes.Ex-cooking causes denaturation. • Denaturation is usually irreversible.Ex-omelet can be prepared from egg but the reversal is not possible. • Careful denaturation is sometimes reversible (Renaturation).Hemoglobin undergoes denaturation in the presence of salicylate.By removal of salicylate ,hemoglobin is renatured. • Denatured protein can’t be crystallized.
  45. 45. Coagulation • Term ‘coagulum’ refers to semi-solid viscous precipitate of protein.irreversible denaturation results in coagulation.coagulation is optimum and requires lowest temperature at isoelectric Ph.Albumins and globulins are coagulable proteins.HEAT COAGULATION TEST IS COMMONLY USED TO DETECT THE PRESENCE OF ALBUMIN IN URINE.