1. The Three-Dimensional Structure of Proteins Chapter 4

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3. Protein Structure Proteins are polymers of amino acids linked by covalent peptide bonds – leads to different conformations Protein’s native conformation – has biological activity

4. Levels of protein structure Primary structure is the sequence of amino acids in a polypeptide chain (covalently linked), from N-terminal end to C-terminal Secondary structure is the arrangement in the space of atoms/localized regions of a polypeptide backbone chain - Repetitive interactions resulting from hydrogen bonding between amide N-H and carbonyl groups of peptide backbone - Side chain conformations of amino acids are not part of secondary structure - Independently folded portions of proteins – domains or super secondary structure e. g., α-helix and β-pleated sheet

5. Levels of proteins structure Tertiary structure is the three-dimensional arrangement of all atoms in the protein, including those in side chains and in any prosthetic groups Quaternary structure includes those proteins which have multiple polypeptide chains called subunits - Mediated by noncovalent interactions such as hydrogen bonds, electrostatic attractions and hydrophobic interactions

6. Primary structure of proteins Primary structure of a protein determines its other levels of structure Determines its properties and functioning A single amino acid substitution – sickle cell anemia Site directed mutagenesis – amino acid residue can be replaced with another amino acid

7. Secondary structure of protein 2˚ of proteins is hydrogen-bonded arrangement of backbone of the protein Two bonds have free rotation: Ramachandran angles 1) Bond between  -carbon and amino nitrogen in residue - Ǿ (phi) angle 2) Bond between the  -carbon and carboxyl carbon of residue – Ψ (psi) angle

8. α-Helix

9. What are the two periodic structures in protein backbones? Coil of the helix is clockwise or right-handed 3.6 amino acids per turn/helix Repeat distance-Pitch- is 5.4Å C=O of each peptide bond is hydrogen bonded to the N-H of the fourth amino acid away All R groups point outward from helix.

10. What are the factors disrupting α-helix? Proline has cyclic structure – does not fit into α- helical structure - Rotation around the bond between the nitrogen and the α-carbon is restricted - α-amino group cannot participate in intrachain hydrogen bonding

11. What are the factors disrupting α-helix? - α-carbon is outside the helix – crowding of side chains and bonding of carbon to other atom than hydrogen – Valine, Isoleucine and Threonine - Strong electrostatic repulsion caused by the proximity of several side chains of like charge, e.g., Lys and Arg or Glu and Asp

12.  -Pleated Sheet Parallel Antiparallel

13. β-sheet C=O----H-N hydrogen bonds are perpendicular to direction of the sheet R groups are alternating – first above and then below the plane

14. Differences α-helix Rodlike structure and involves one polypeptide chain Hydrogen bonding is parallel to α-helix within backbone β-sheet Pleated structure and involves one or more polypeptide chain Hydrogen bonding is perpendicular to direction of protein chain

15.  -bulge- a common no repetitive irregular 2˚ motif in anti-parallel structure  -Pleated Sheet

16. Why is Glycine frequently found in reverse turns? Reverse turns make transition from secondary structure to another form Single hydrogen of side chain prevents crowding Proline (cyclic structure) has perfect geometry

17. Schematic Diagrams of Supersecondary Structures

18. What are the Supersecondary structures and domains? Combinations of α-helix and β-pleated sheet – Supersecondary structures βαβ unit: Two parallel strands of β-sheet are connected by a stretch of α-helix αα unit: Two antiparallel α-helices (helix-turn- helix) β-meander: an antiparallel sheet formed by series of tight reverse turns connecting stretches of polypeptide chain

19. What are the Supersecondary structures and domains? Greek key: an antiparallel sheet formed when polypeptide chain doubles back on itself β-barrel : when β-sheets are extensive enough to fold back on themselves - β-meander or Greek key can be found in β-barrel in tertiary structure of proteins (figure 4.10) - Motifs are repetitive supersecondary structures (figure 4.9)

20. Collagen Triple Helix (figure 4.11) Consists of three polypeptide chains wrapped around each other in a ropelike twist – triple helix – Tropocollagen- not α helix Each chain – repeating sequence of three amino acids, X- Pro-Gly or X-Hyp-Gly 30% of aa in each chain are Pro and Hyp. Hydroxylysine is also found Three strands held by hydrogen bonding – hydroxyproline and hydroxylysine Intramolecular and intermolecular linking by covalent bonds – formed by reactions of lysine and histidine aa

21. Collagen Triple Helix Linking between lysine and histidine residues increases with age – meat in older animals is tougher than younger animals Lack of hydroxylation of proline to hydroxyproline – makes collagen less stable Scurvy – result of fragile collagen Vitamin C prescribed for Scurvy

22. Protein Conformation: Fibrous and globular proteins Fibrous proteins consist of parallel long fibers or large sheets consist of parallel long fibers or large sheets mechanically strong mechanically strong insoluble in water and dilute salt solutions insoluble in water and dilute salt solutions play important structural roles in nature play important structural roles in nature Examples: Keratin of hair and wool, collagen etc Examples: Keratin of hair and wool, collagen etc Globular proteins folded to more or less spherical shape All have α helices and β sheets Soluble in water and salt solutions Tertiary and quaternary structures are complex

23. Forces involved in tertiary structure of proteins Noncovalent interactions -Hydrogen bonding between polar side chains, eg: Serine and threonine -Non polar side chains – hydrophobic interactions. Eg: Valine and Isoleucine -Electrostatic attraction between oppositely charged groups. Eg: Lysine and Glutamine Covalent interactions – disulfide (-S-S) bonds between side chains of cysteines Myoglobin and hemoglobin – no disulfide bonds Trypsin and chymotrypsin – have disulfide bonds

24. Forces That Stabilize Protein Structure

25. How can three dimensional structure of protein be determined? X-ray crystallography determines tertiary structure Perfect crystals of some proteins can be grown under carefully controlled conditions Crystal exposure to beam of X-rays – diffraction pattern is produced on a photographic plate or a radiation counter Heavier atoms scatter more effectively than the other Scattered X-rays from individual atoms can reinforce or cancel each other – gives rise to characteristic pattern for each type of molecule

26. X-ray crystallography Series of diffraction patterns taken from several angles – needed information to determine tertiary structure Extracted via Fourier series – a mathematical analysis Thousands of such calculations – determine structure of protein – performed by computer

27. Nuclear magnetic resonance spectroscopy (2D) Protein samples are present in aqueous solution (small quantities in milligrams) Determines protein structure based on distance between hydrogen atoms Computer analysis- Fourier series

28. Denaturation and Renaturation Unfolding - denaturation of protein Refolding– recovery of unfolded protein

29. Denaturation of proteins Denaturation – heat - High or low extremes of pH - Detergents (SDS) – disrupt electrostatic (hydrophobic) interactions - Urea or guanidine disrupt hydrogen bonding - β-mercaptoethanol – reduces disulfide bridges/bonds

30. Quaternary structure of protein The association of polypeptide monomers into multisubunit proteins - dimers, trimers and tetramers - Noncovalent interactions present – electrostatic, hydrophobic interactions and hydrogen bonding - Many multisubunit proteins – allosteric effects

31. Differences between Myo and Hemoglobin Myoglobin-single polypeptide chain with 153 aa, 8 α-helical regions and prosthetic group-heme Hemoglobin – two α (141 aa) and two β chains (146 aa) One molecule of oxygen binds to one molecule of Myo. Four molecules of oxygen bind to one molecule of hemo

32. Why does oxygen have imperfect binding to heme group in Myoglobin? CO (25,000) and O2 can bind to heme Blocked by His E7 Heme must release O2 In absence of oxygen carrying proteins – iron of heme group can be oxidized to Fe (III)-will not bind to O2 No positive cooperativity

33. What is positive cooperativity/cooperative binding? One oxygen binds – it makes subsequent binding of oxygen molecules easy Graph 4.22 – oxygen binding curve in myoglobin is hyperbolic and in hemoglobin is sigmoidal H +, CO 2, Cl -, and 2,3-bisphosphoglycerate (BPG) affect the ability of Hb to bind and transport oxygen

34. How does Hemoglobin work? Affinity for oxygen is controlled by several factors – oxygen pressure and pH When pH drops or oxygen pressure is low – hemoglobin tend to release more oxygen and viceversa-binds to oxygen

35. Oxygenated and deoxygenated hemoglobin Hemoglobin has different quaternary structures in unbound (deoxygenated) and bound (oxygenated) β-chains are closer in oxygenated hemoglobin than deoxygenated hemoglobin Different crystal structures

36. Protein folding dynamics Prediction of tertiary structure of protein – possible. Biochemistry + Computers – Bioinformatics Prediction – sequence homology

37. Hydrophillic and Hydrophobic interactions

38. Why is correct folding of proteins important? Correctly folded proteins – soluble in aqueous environment and attached to membranes Incorrectly folded proteins – form aggregates with other proteins (figure 4.35) Several neurodegenerative disorders – Alzheimer’s, Parkinson’s and Huntington’s diseases Prion disease – mad cow disease

39. What are Chaperones? Chaperones are special proteins Aid in timely and correct folding of proteins Hsp 70 - first discovered chaperones in E.coli Exist in all organisms – prokaryotes to humans

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