Proteins chp-4-bioc-361-version-oct-2012b


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Biochemistry - Campbell 6e - Enzymes. Based on UAEU Chem 361 syllabus.

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Proteins chp-4-bioc-361-version-oct-2012b

  1. 1. Mary K. Campbell Shawn O. Farrell Chapter FourThe Three-Dimensional Structure of Proteins Paul D. Adams • University of Arkansas
  2. 2. Protein Structure• Many conformations are possible for proteins: • Due to flexibility of amino acids linked by peptide bonds• At least one major conformations has biological activity, and hence is considered the protein’s native conformation
  3. 3. Levels of Protein Structure1° structure: the sequence of amino acids in a polypeptide chain, read from the N-terminal end to the C-terminal end• 2° structure: the ordered 3-dimensional arrangements (conformations) in localized regions of a polypeptide chain; refers only to interactions of the peptide backbone • e. g., α-helix and β-pleated sheet• 3˚ structure: 3-D arrangement of all atoms• 4˚ structure: arrangement of monomer subunits with respect to each other
  4. 4. 1˚ Structure• The 1˚ sequence of proteins determines its 3-D conformation• Changes in just one amino acid in sequence can alter biological function, e.g. hemoglobin associated with sickle-cell anemia• Determination of 1˚ sequence is routine biochemistry lab work (See Ch. 5).
  5. 5. 2˚ Structure• 2˚ of proteins is hydrogen-bonded arrangement of backbone of the protein• Two bonds have free rotation: 1) Bond between α-carbon and amino nitrogen in residue 2) Bond between the α-carbon and carboxyl carbon of residue• See Figure 4.1
  6. 6. α-Helix• Coil of the helix is clockwise or right-handed• There are 3.6 amino acids per turn• Repeat distance is 5.4Å• Each peptide bond is s-trans and planar• C=O of each peptide bond is hydrogen bonded to the N-H of the fourth amino acid away• C=O----H-N hydrogen bonds are parallel to helical axis• All R groups point outward from helix
  7. 7. α-Helix (Cont’d)
  8. 8. α-Helix (Cont’d)• Several factors can disrupt an α-helix • proline creates a bend because of (1) the restricted rotation due to its cyclic structure and (2) its α-amino group has no N-H for hydrogen bonding • strong electrostatic repulsion caused by the proximity of several side chains of like charge, e.g., Lys and Arg or Glu and Asp • steric crowding caused by the proximity of bulky side chains, e.g., Val, Ile, Thr
  9. 9. β-Pleated Sheet• Polypeptide chains lie adjacent to one another; may be parallel or antiparallel• R groups alternate, first above and then below plane• Each peptide bond is s-trans and planar• C=O and N-H groups of each peptide bond are perpendicular to axis of the sheet• C=O---H-N hydrogen bonds are between adjacent sheets and perpendicular to the direction of the sheet
  10. 10. β-Pleated Sheet (Cont’d)
  11. 11. Structures of Reverse Turns• Glycine found in reverse turns• Spatial (steric) reasons• Polypeptide changes direction• Proline also encountered in reverse turns. Why?
  12. 12. α-Helices and β-Sheets• Supersecondary structures: the combination of α- and β-sections, as for example • βαβ unit: two parallel strands of β-sheet connected by a stretch of α-helix • αα unit: two antiparallel α-helices • β -meander: an antiparallel sheet formed by a series of tight reverse turns connecting stretches of a polypeptide chain • Greek key: a repetitive supersecondary structure formed when an antiparallel sheet doubles back on itself • β -barrel: created when β-sheets are extensive enough to fold back on themselves
  13. 13. Schematic Diagrams of SupersecondaryStructures
  14. 14. Fibrous Proteins• Fibrous proteins: contain polypeptide chains organized approximately parallel along a single axis. They • consist of long fibers or large sheets • tend to be mechanically strong • are insoluble in water and dilute salt solutions • play important structural roles in nature• Examples are • keratin of hair and wool • collagen of connective tissue of animals including cartilage, bones, teeth, skin, and blood vessels
  15. 15. Globular Proteins• Globular proteins: proteins which are folded to a more or less spherical shape • they tend to be soluble in water and salt solutions • most of their polar side chains are on the outside and interact with the aqueous environment by hydrogen bonding and ion-dipole interactions • most of their nonpolar side chains are buried inside • nearly all have substantial sections of α-helix and β- sheet
  16. 16. Comparison of Shapes of Fibrous andGlobular Proteins
  17. 17. 3˚ Structure• The 3-dimensional arrangement of atoms in the molecule.• In fibrous protein, backbone of protein does not fall back on itself, it is important aspect of 3˚ not specified by 2˚ structure.• In globular protein, more information needed. 3k structure allows for the determination of the way helical and pleated-sheet sections fold back on each other.• Interactions between side chains also plays a role.
  18. 18. Forces in 3˚ Structure• Noncovalent interactions, including • hydrogen bonding between polar side chains, e.g., Ser and Thr • hydrophobic interaction between nonpolar side chains, e.g., Val and Ile • electrostatic attraction between side chains of opposite charge, e.g., Lys and Glu • electrostatic repulsion between side chains of like charge, e.g., Lys and Arg, Glu and Asp• Covalent interactions: Disulfide (-S-S-) bonds between side chains of cysteines
  19. 19. Forces That Stabilize Protein Structure
  20. 20. 3° and 4° Structure• Tertiary (3°) structure: the arrangement in space of all atoms in a polypeptide chain • it is not always possible to draw a clear distinction between 2° and 3° structure• Quaternary (4°) structure: the association of polypeptide chains into aggregations• Proteins are divided into two large classes based on their three-dimensional structure • fibrous proteins • globular proteins
  21. 21. Determination of 3° Structure• X-ray crystallography • uses a perfect crystal; that is, one in which all individual protein molecules have the same 3D structure and orientation • exposure to a beam of x-rays gives a series diffraction patterns • information on molecular coordinates is extracted by a mathematical analysis called a Fourier series• 2-D Nuclear magnetic resonance • can be done on protein samples in aqueous solution
  22. 22. X-Ray and NMR Data High resolution method to determine 3˚ structure of proteins (from crystal) Determines solution structure Diffraction pattern produced by electrons Structural info. Gained from scattering X-rays determining distances between Series of patterns taken at different nuclei that aid in structure angles gives structural information determination
  23. 23. Myoglobin• A single polypeptide chain of 153 amino acids• A single heme group in a hydrophobic pocket• 8 regions of α-helix; no regions of β-sheet• Most polar side chains are on the surface• Nonpolar side chains are folded to the interior• Two His side chains are in the interior, involved with interaction with the heme group• Fe(II) of heme has 6 coordinates sites; 4 interact with N atoms of heme, 1 with N of a His side chain, and 1 with either an O2 molecule or an N of the second His side chain
  24. 24. The Structure of Myoglobin
  25. 25. Oxygen Binding Site of Myoglobin
  26. 26. Denaturation• Denaturation: the loss of the structural order (2°, 3°, 4°, or a combination of these) that gives a protein its biological activity; that is, the loss of biological activity• Denaturation can be brought about by • heat • large changes in pH, which alter charges on side chains, e.g., -COO- to -COOH or -NH3+ to -NH2 • detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions • urea or guanidine, which disrupt hydrogen bonding • mercaptoethanol, which reduces disulfide bonds
  27. 27. Denaturation of a Protein
  28. 28. Denaturation and Refolding inRibonucleaseSeveral ways to denatureproteins• Heat• pH• Detergents• Urea• Guanadine hydrochloride
  29. 29. Quaternary Structure• Quaternary (4°) structure: the association of polypepetide monomers into multisubunit proteins • dimers • trimers • tetramers• Noncovalent interactions • electrostatics, hydrogen bonds, hydrophobic
  30. 30. Oxygen Binding of Hemoglobin (Hb)• A tetramer of two α-chains (141 amino acids each) and two β-chains (153 amino acids each); α2β2• Each chain has 1 heme group; hemoglobin can bind up to 4 molecules of O2• Binding of O2 exhibited by positive cooperativity; when one O2 is bound, it becomes easier for the next O2 to bind• The function of hemoglobin is to transport oxygen• The structure of oxygenated Hb is different from that of unoxygenated Hb• H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG) affect the ability of Hb to bind and transport oxygen
  31. 31. Structure of Hemoglobin
  32. 32. Conformation Changes That Accompany Hb Function• Structural changes occur during binding of small molecules• Characteristic of allosteric behavior• Hb exhibits different 4˚ structure in the bound and unbound oxygenated forms• Other ligands are involved in cooperative effect of Hb can affect protein’s affinity for O2 by altering structure
  33. 33. Oxy- and Deoxyhemoglobin
  34. 34. Primary Structure DeterminationHow is 1˚ structure determined?1) Determine which amino acids are present (amino acid analysis)2) Determine the N- and C- termini of the sequence (a.a sequencing), and the Internal Residues3) Determine the sequence of smaller peptide fragments (most proteins > 100 a.a)4) Some type of cleavage into smaller units necessary
  35. 35. Primary Structure Determination
  36. 36. Protein CleavageProtein cleaved at specific sites by:1) Enzymes- Trypsin, Chymotrypsin, Carboxypeptidases (C- terminus)2) Chemical reagents- Cyanogen bromide, cleaves at Methionine;- PITC, cleaves from N-terminus (Edman Degradation)- Hydrazine, cleaves from C-terminusEnzymes which cleaves Internal Residues:Trypsin- Cleaves @ C-terminal of (+) charged side chains (basic amino acid)Chymotrypsin- Cleaves @ C-terminal of aromatics
  37. 37. Peptide Digestion
  38. 38. Cleavage by CnBrCleaves @ C-terminal of INTERNAL methionines
  39. 39. Determining Protein SequenceAfter cleavage, mixture of peptide fragments produced.• Can be separated by HPLC or other chromatographic techniques• Use different cleavage reagents to help in 1˚ determination
  40. 40. Peptide Sequencing• Can be accomplished by Edman Degradation• Relatively short sequences (30-40 amino acids) can be determined quickly• So efficient, today N-/C-terminal residues usually not done by enzymatic/chemical cleavage
  41. 41. Peptide Sequencing