Campbell6e lecture ch4


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Campbell6e lecture ch4

  1. 1. Chapter Four The Three-Dimensional Structure of Proteins
  2. 2. Protein Structure <ul><li>Many conformations are possible for proteins: </li></ul><ul><ul><li>Due to flexibility of amino acids linked by peptide bonds </li></ul></ul><ul><li>• At least one major conformations has biological activity, and hence is considered the protein’s native conformation </li></ul>
  3. 3. Levels of Protein Structure <ul><li>1° structure: the sequence of amino acids in a polypeptide chain, read from the N-terminal end to the C-terminal end </li></ul><ul><li>2° structure: the ordered 3-dimensional arrangements (conformations) in localized regions of a polypeptide chain; refers only to interactions of the peptide backbone </li></ul><ul><ul><li>e. g.,  -helix and  -pleated sheet </li></ul></ul><ul><li>• 3˚ structure: 3-D arrangement of all atoms </li></ul><ul><li>• 4˚ structure: arrangement of monomer subunits with respect to each other </li></ul>
  4. 4. 1˚ Structure <ul><li>The 1˚ sequence of proteins determines its 3-D conformation </li></ul><ul><li>Changes in just one amino acid in sequence can alter biological function, e.g. hemoglobin associated with sickle-cell anemia </li></ul><ul><li>Determination of 1˚ sequence is routine biochemistry lab work (See Ch. 5). </li></ul>
  5. 5. 2˚ Structure <ul><li>• 2˚ of proteins is hydrogen-bonded arrangement of backbone of the protein </li></ul><ul><li>• Two bonds have free rotation: </li></ul><ul><ul><li>Bond between  -carbon and amino nitrogen in residue </li></ul></ul><ul><ul><li>Bond between the  -carbon and carboxyl carbon of residue </li></ul></ul><ul><li>• See Figure 4.1 </li></ul>
  6. 6.  -Helix <ul><li>Coil of the helix is clockwise or right-handed </li></ul><ul><li>There are 3.6 amino acids per turn </li></ul><ul><li>Repeat distance is 5.4Å </li></ul><ul><li>Each peptide bond is s-trans and planar </li></ul><ul><li>C=O of each peptide bond is hydrogen bonded to the N-H of the fourth amino acid away </li></ul><ul><li>C=O----H-N hydrogen bonds are parallel to helical axis </li></ul><ul><li>All R groups point outward from helix </li></ul>
  7. 7.  -Helix (Cont’d)
  8. 8.  -Helix (Cont’d) <ul><li>Several factors can disrupt an  -helix </li></ul><ul><ul><li>proline creates a bend because of (1) the restricted rotation due to its cyclic structure and (2) its  -amino group has no N-H for hydrogen bonding </li></ul></ul><ul><ul><li>strong electrostatic repulsion caused by the proximity of several side chains of like charge, e.g., Lys and Arg or Glu and Asp </li></ul></ul><ul><ul><li>steric crowding caused by the proximity of bulky side chains, e.g., Val, Ile, Thr </li></ul></ul>
  9. 9.  -Pleated Sheet <ul><li>Polypeptide chains lie adjacent to one another; may be parallel or antiparallel </li></ul><ul><li>R groups alternate, first above and then below plane </li></ul><ul><li>Each peptide bond is s-trans and planar </li></ul><ul><li>C=O and N-H groups of each peptide bond are perpendicular to axis of the sheet </li></ul><ul><li>C=O---H-N hydrogen bonds are between adjacent sheets and perpendicular to the direction of the sheet </li></ul>
  10. 10.  -Pleated Sheet (Cont’d)
  11. 11. <ul><li> -bulge - a common nonrepetive irregular 2˚ motif in anti-parallel structure </li></ul> -Pleated Sheet (Cont’d)
  12. 12. <ul><li>• Glycine found in reverse turns </li></ul><ul><li>• Spatial (steric) reasons </li></ul><ul><li>• Polypeptide changes direction </li></ul><ul><li>• Proline also encountered in reverse turns. Why? </li></ul>Structures of Reverse Turns
  13. 13.  -Helices and  -Sheets <ul><li>Supersecondary structures : the combination of  - and  -sections, as for example </li></ul><ul><ul><li> unit : two parallel strands of  -sheet connected by a stretch of  -helix </li></ul></ul><ul><ul><li> unit : two antiparallel  -helices </li></ul></ul><ul><ul><li> -meander : an antiparallel sheet formed by a series of tight reverse turns connecting stretches of a polypeptide chain </li></ul></ul><ul><ul><li>Greek key : a repetitive supersecondary structure formed when an antiparallel sheet doubles back on itself </li></ul></ul><ul><ul><li> -barrel : created when  -sheets are extensive enough to fold back on themselves </li></ul></ul>
  14. 14. Schematic Diagrams of Supersecondary Structures
  15. 15. Collagen Triple Helix <ul><li>Consists of three polypeptide chains wrapped around each other in a ropelike twist to form a triple helix called tropocollagen; MW approx. 300,000 </li></ul><ul><li>30% of amino acids in each chain are Pro and Hyp (hydroxyproline); hydroxylysine also occurs </li></ul><ul><li>Every third position is Gly and repeating sequences are X-Pro-Gly and X-Hyp-Gly </li></ul><ul><li>Each polypeptide chain is a helix but not an  -helix </li></ul><ul><li>The three strands are held together by hydrogen bonding involving hydroxyproline and hydroxylysine </li></ul><ul><li>With age, collagen helices become cross linked by covalent bonds formed between Lys and His residues </li></ul>
  16. 16. Fibrous Proteins <ul><li>Fibrous proteins : contain polypeptide chains organized approximately parallel along a single axis. They </li></ul><ul><ul><li>consist of long fibers or large sheets </li></ul></ul><ul><ul><li>tend to be mechanically strong </li></ul></ul><ul><ul><li>are insoluble in water and dilute salt solutions </li></ul></ul><ul><ul><li>play important structural roles in nature </li></ul></ul><ul><li>Examples are </li></ul><ul><ul><li>keratin of hair and wool </li></ul></ul><ul><ul><li>collagen of connective tissue of animals including cartilage, bones, teeth, skin, and blood vessels </li></ul></ul>
  17. 17. Globular Proteins <ul><li>Globular proteins: proteins which are folded to a more or less spherical shape </li></ul><ul><ul><li>they tend to be soluble in water and salt solutions </li></ul></ul><ul><ul><li>most of their polar side chains are on the outside and interact with the aqueous environment by hydrogen bonding and ion-dipole interactions </li></ul></ul><ul><ul><li>most of their nonpolar side chains are buried inside </li></ul></ul><ul><ul><li>nearly all have substantial sections of  -helix and  -sheet </li></ul></ul>
  18. 18. Comparison of Shapes of Fibrous and Globular Proteins
  19. 19. 3˚ Structure <ul><li>The 3-dimensional arrangement of atoms in the molecule. </li></ul><ul><li>In fibrous protein, backbone of protein does not fall back on itself, it is important aspect of 3˚ not specified by 2˚ structure. </li></ul><ul><li>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. </li></ul><ul><li>Interactions between side chains also plays a role. </li></ul>
  20. 20. Forces in 3˚ Structure <ul><li>Noncovalent interactions , including </li></ul><ul><ul><li>hydrogen bonding between polar side chains, e.g., Ser and Thr </li></ul></ul><ul><ul><li>hydrophobic interaction between nonpolar side chains, e.g., Val and Ile </li></ul></ul><ul><ul><li>electrostatic attraction between side chains of opposite charge, e.g., Lys and Glu </li></ul></ul><ul><ul><li>electrostatic repulsion between side chains of like charge, e.g., Lys and Arg, Glu and Asp </li></ul></ul><ul><li>Covalent interactions : Disulfide (-S-S-) bonds between side chains of cysteines </li></ul>
  21. 21. Forces That Stabilize Protein Structure
  22. 22. 3° and 4° Structure <ul><li>Tertiary (3°) structure : the arrangement in space of all atoms in a polypeptide chain </li></ul><ul><ul><li>it is not always possible to draw a clear distinction between 2° and 3° structure </li></ul></ul><ul><li>Quaternary (4°) structure : the association of polypeptide chains into aggregations </li></ul><ul><li>Proteins are divided into two large classes based on their three-dimensional structure </li></ul><ul><ul><li>fibrous proteins </li></ul></ul><ul><ul><li>globular proteins </li></ul></ul>
  23. 23. Determination of 3° Structure <ul><li>X-ray crystallography </li></ul><ul><ul><li>uses a perfect crystal; that is, one in which all individual protein molecules have the same 3D structure and orientation </li></ul></ul><ul><ul><li>exposure to a beam of x-rays gives a series diffraction patterns </li></ul></ul><ul><ul><li>information on molecular coordinates is extracted by a mathematical analysis called a Fourier series </li></ul></ul><ul><li>2-D Nuclear magnetic resonance </li></ul><ul><ul><li>can be done on protein samples in aqueous solution </li></ul></ul>
  24. 24. High resolution method to determine 3˚ structure of proteins (from crystal) Diffraction pattern produced by electrons scattering X-rays Series of patterns taken at different angles gives structural information Determines solution structure Structural info. Gained from determining distances between nuclei that aid in structure determination X-Ray and NMR Data
  25. 25. Myoglobin <ul><li>A single polypeptide chain of 153 amino acids </li></ul><ul><li>A single heme group in a hydrophobic pocket </li></ul><ul><li>8 regions of  -helix; no regions of  -sheet </li></ul><ul><li>Most polar side chains are on the surface </li></ul><ul><li>Nonpolar side chains are folded to the interior </li></ul><ul><li>Two His side chains are in the interior, involved with interaction with the heme group </li></ul><ul><li>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 O 2 molecule or an N of the second His side chain </li></ul>
  26. 26. The Structure of Myoglobin
  27. 27. Oxygen Binding Site of Myoglobin
  28. 28. Denaturation <ul><li>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 </li></ul><ul><li>Denaturation can be brought about by </li></ul><ul><ul><li>heat </li></ul></ul><ul><ul><li>large changes in pH, which alter charges on side chains, e.g., -COO - to -COOH or -NH  + to -NH  </li></ul></ul><ul><ul><li>detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions </li></ul></ul><ul><ul><li>urea or guanidine, which disrupt hydrogen bonding </li></ul></ul><ul><ul><li>mercaptoethanol, which reduces disulfide bonds </li></ul></ul>
  29. 29. Denaturation of a Protein
  30. 30. Several ways to denature proteins • Heat • pH •   Detergents • Urea • Guanadine hydrochloride Denaturation and Refolding in Ribonuclease
  31. 31. Quaternary Structure <ul><li>Quaternary (4°) structure : the association of polypepetide monomers into multisubunit proteins </li></ul><ul><ul><li>dimers </li></ul></ul><ul><ul><li>trimers </li></ul></ul><ul><ul><li>tetramers </li></ul></ul><ul><li>Noncovalent interactions </li></ul><ul><ul><li>electrostatics, hydrogen bonds, hydrophobic </li></ul></ul>
  32. 32. Oxygen Binding of Hemoglobin (Hb) <ul><li>A tetramer of two  -chains (141 amino acids each) and two  -chains (153 amino acids each);  2  2 </li></ul><ul><li>Each chain has 1 heme group; hemoglobin can bind up to 4 molecules of O 2 </li></ul><ul><li>Binding of O 2 exhibited by positive cooperativity ; when one O 2 is bound, it becomes easier for the next O 2 to bind </li></ul><ul><li>The function of hemoglobin is to transport oxygen </li></ul><ul><li>The structure of oxygenated Hb is different from that of unoxygenated Hb </li></ul><ul><li>H + , CO 2 , Cl - , and 2,3-bisphosphoglycerate (BPG) affect the ability of Hb to bind and transport oxygen </li></ul>
  33. 33. Structure of Hemoglobin
  34. 34. Conformation Changes That Accompany Hb Function <ul><li>• Structural changes occur during binding of small molecules </li></ul><ul><li>• Characteristic of allosteric behavior </li></ul><ul><li>• Hb exhibits different 4˚ structure in the bound and unbound oxygenated forms </li></ul><ul><li>• Other ligands are involved in cooperative effect of Hb can affect protein’s affinity for O 2 by altering structure </li></ul>
  35. 35. Oxy- and Deoxyhemoglobin
  36. 36. Protein Folding Dynamics <ul><li>•   Can 3˚ structure of protein be predicted? Yes , within limitations </li></ul><ul><li>• The integration of biochemistry and computing has led to bioinformatics </li></ul><ul><li>• Protein structure prediction is one of the principal application of bioinformatics </li></ul><ul><li>• First step to predict protein structure is to search for sequence homology </li></ul>
  37. 37. Predicting Protein Structure
  38. 38. Hydrophobic Interactions <ul><li>• Hydrophobic interactions are major factors in protein folding </li></ul><ul><li>• Folds so that nonpolar hydrophobic side chains tend to be on inside away from water, and polar side chains on outside accessible to aqueous environment </li></ul><ul><li>• Hydrophobic interactions are spontaneous </li></ul>
  39. 39. Hydrophobic and Hydrophilic Interactions in Proteins
  40. 40. Protein Folding Chaperones <ul><li>In the protein-dense environment of a cell, proteins may begin to fold incorrectly or may associate with other proteins before folding is completed </li></ul><ul><li>Special proteins called chaperones aid in the correct and timely folding of many proteins </li></ul><ul><li>hsp70 were the first chaperone proteins discovered </li></ul><ul><li>Chaperones exist in organisms from prokaryotes to humans </li></ul>
  41. 41. AMINO ACID SEQUENCING <ul><li>N-TERMINUS </li></ul><ul><li>Edman Degradation: uses phenylisothiocyanate (PITC); cycle repeated </li></ul><ul><li>C-TERMINUS : uses exopeptidases (carboxypeptidase), and Hydrazine </li></ul><ul><li>INTERNAL RESIDUES: </li></ul><ul><li>Trypsin which cleaves –CONH donated by basic amino acids; </li></ul><ul><li>Chymotrypsin which cleaves –CONH donat4ed by aromatic amino acids; </li></ul><ul><li>Cyanogen Bromide which cleaves –CONH donated by methionine </li></ul>