1. Advanced Biochemistry 高等生化學 The Three-Dimensional Structure of Proteins 陳威戎

2. Perhaps the more remarkable features of [myoglobin] are its complexity and its lack of symmetry. The arrangement seems to be almost totally lacking in the kind of regularities which one instinctively anticipates, and it is more complicated than has been predicted by any theory of protein structure. - J. Kendrew, article in Naure, 1958 Preface Max Perutz, 1941-2002 John Kendrew, 1917-1997

3. 1. The three-dimensional structure of a protein is determined by its amino acid sequence. 2. The function of a protein depends on its structure. 3. An isolated protein exists in one or few stable structural forms. 4. The most important forces stabilizing the protein structures are noncovalent interactions. 5. Common structural patterns that help us organize our understanding of protein architecture. Five themes to emphasize in this chapter

4. Structure of the enzyme chymotrypsin, a globular protein

5. Protein Data Bank, PDB (www.rcsb.org/pdb) PDB ID: four-character identifier (ex: 6GCH) Free molecular graphic programs: RasMol, Chime, Swiss-Pdb Viewer Internet resources for protein structures

6. Protein Data Bank (PDB) Research Collaboration for Structural Bioinformatics http://www.rcsb.org/pdb/

10. 1.Overview of Protein Structure 2.Protein Secondary Structure 3.Protein Tertiary and Quaternary Structures 4.Protein Denaturation and Folding Three-Dimensional Structure of Proteins

11. 1. A protein’s conformation is stabilized largely by weak interactions. 2. The peptide bond is rigid and planar. Overview of Protein Structure

12. 1. A protein’s conformation is stabilized largely by weak interactions. Stability: the tendency to maintain a native conformation Unfolded state: high degree of conformational entropy Two simple rules: (1) Hydrophobic residues are largely buried in the protein interior, away from water. (2) The number of hydrogen bonds within the protein is maximized.

13. 2. The peptide bond is rigid and planar

16. Ramachandran plot for L-Ala residues

17. 1.Overview of Protein Structure 2.Protein Secondary Structure 3.Protein Tertiary and Quaternary Structures 4.Protein Denaturation and Folding Three-Dimensional Structure of Proteins

18. 1. The  helix is a common protein secondary structure. 2. Amino acid sequence affects  helix stability. 3. The  conformation organizes polypeptide chains into sheets. 4.  turns are common in proteins. 5. Common secondary structures have characteristic bond angles and amino acid content. Protein Secondary Structure

19. 1. The  helix is a common protein secondary structure

20. Knowing the right hand from the left

21. Five constraints affect the stability of an  helix 1.The electrostatic repulsion (or attraction) between successive amino acid residues with charged R groups 2.The bulkiness of adjacent R groups 3.The interactions between R groups spaced three (or four) residues apart 4.The occurrence of Pro and Gly residues 5. The interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the  helix

22. 2. Amino acid sequence affects  helix stability Interactions between R groups of amino acids three residues apart in an  helix

23. 2. Amino acid sequence affects a helix stability Helix dipole

24. 3. The  conformation organize polypeptide chains into sheets

26. 4.  turns are common in proteins

28. 5. Common secondary structures have characteristic bond angles and amino acid content

31. 1.Overview of Protein Structure 2.Protein Secondary Structure 3.Protein Tertiary and Quaternary Structures 4.Protein Denaturation and Folding Three-Dimensional Structure of Proteins

32. 1. Fibrous proteins are adapted for a structural function. 2. Structural diversity reflects functional diversity in globular proteins. 3. Myoglobin provided early clues about the complexity of globular protein structure. 4. Globular proteins have a variety of tertiary structures. 5. Analysis of many globular proteins reveals common structural patterns. 6. Protein motifs are the basis for protein structural classification. 7. Protein quaternary structures range from simple dimers to large complexes. 8. There are limits to the size of proteins. Protein Tertiary and Quaternary Structures

33. Fibrous proteins vs. globular proteins Fibrous proteinsGlobular proteins Polypeptide chain Long strands or sheets Spherical or globular shape Secondary structures Single typeSeveral types Protein Functions Support, shape and external protection Enzymes and regulatory proteins

34. 1. Fibrous proteins are adapted for a structural function

35. 1. Evolved for strength. 2. Found in mammals, constitutes: hair, wool, nails, claws, quills, horns, hooves, and much of the outer layer of skin. 3. Part of intermediate filament (IF) proteins. 4. Right-handed  -helix. 5. Rich in hydrophobic residues: Ala, Val, Leu, Ile, Met, and Phe. 6. Strength enhanced by covalent cross-links.  -keratin

36. Structure of hair  -keratin

37. Cross section of a hair

38. Permanent waving is biochemical Engineering

39. 1. Evolved to provide strength. 2. Found in connective tissues such as: tendons, cartilage, the organic matrix of bone, and the cornea of the eye. 3. Left-handed triple helix, three a.a. per turn. 4. Three supertwisted polypeptides,  chains. 5. Typically contain: 35% Gly, 11% Ala, 21% Pro and 4-Hyp (4-hydroxyproline). 6. Repeating tripeptide unit: Gly-Pro-4-Hyp Collagen

40. Structure of collagen

41. Structure of collagen fibrils

42. 1. Fibroin, the protein of silk, is produced by insects and spiders. 2. Predominantly in  conformation. 3. Soft and flexible. 4. Stabilized by extensive hydrogen bonding. 5. Rich in Ala and Gly. Silk Fibroin

43. Structure of silk

44. Strands of fibroin emerge from the spinnerets of a spider

45. Why sailors, explorers, and college students should eat their fresh fruits and vegetables! Scurvy: caused by lack of vitamin C small hemorrhages caused by fragile blood vessels, tooth loss, poor wound healing, reopening of old wounds, bone pain and degeneration, and eventually heart failure. Vitamin C: required for hydroxylation of proline and lysine in collagen Scurvy vs. Vitamin C (Ascorbic acid)

46. Vitamin C (L-ascorbic acid) is a white, odorless, crystalline powder. It is freely soluble in water. Recommended daily allowance: 60 mg (USA) Scurvy vs. Vitamin C (Ascorbic acid)

47. Repeating tripeptide unit in collagen: Gly-Pro-4-Hyp: Tm= 69 ℃ Gly-Pro-Pro: Tm= 41 ℃ Scurvy vs. Vitamin C (Ascorbic acid)

48. Prolyl 4-hydroxylase:  2  2 tetramer, each  sununit contains one atom of nonheme iron (Fe 2+ ) Scurvy vs. Vitamin C (Ascorbic acid)

49. 2. Structural diversity reflects functional diversity in globular proteins

50. 3. Myoglobin provided early clues about the complexity of globular protein structure Tertiary structure of sperm whale myoglobin

51. The heme group

52. 4. Globular proteins have a variety of tertiary structures

54. Methods for determining the three-dimensional structure of a protein: X-ray diffraction

55. Protocols: 1. Protein over-expression and purification 2. Protein crystallization 3. X-ray diffraction 4. Phase determination and electron density maps 5. Model building and refinement Advantages: 1. Best resolution 2. No size limitation (in contrast to NMR) Limitations: Technically very challenging to make crystals of proteins. (heterogeneous samples, membrane proteins, protein complexes)

56. Methods for determining the three-dimensional structure of a protein: Nuclear magetic resonance, NMR

57. Protocols: 1. A concentrated aqueous protein sample (0.2-1 mM, 6-30 mg/mL) labeled with 13 C and/or 15 N is placed in a large magnet. 2. An external magnetic field is applied; 13 C and 15 N nuclei will undergo precession (spinning like a cone) with a frequency that depends on the external environment 3. From these frequencies, computer determines the through-bond (J coupling) and through-space (NOE) constants between every pair of NMR-active nuclei. 4. These values provide a set of estimates of distances between specific pairs of atoms, called "constraints“ 5. Build a model for the structure that is consistent with the set of constraints

60. Methods for determining the three-dimensional structure of a protein: Nuclear magetic resonance, NMR Advantages: 1. Native like conditions – sample is hydrated, not in a crystal lattice 2. Can get dynamic information – observe conformational changes 3. Can look at relative disorder of specific regions of a protein – can see if a loop is static or flexible over time Limitations: 1. Not as high resolution as x-ray 2. Require a lot of protein to get a good signal 3. Require very concentrated samples (can get insoluble aggregates) 4. Limit on protein size measurable, since molecule must tumble rapidly to give sharp peaks. Typically, proteins must be <30kD.

61. 5. Analysis of many globular proteins reveals common structural patterns 1.The three-dimensional structure of a typical globular protein can be considered an assemblage of polypeptide segments in the a-helix and b-sheet conformations. 2.Supersecondary structures: motifs, folds Stable arrangements of several elements of secondary structure and the connections between them. 3. Polypeptides with more than a few hundred amino acid residues often fold into two or more stable, globular units called domains.

62. 5. Analysis of many globular proteins reveals common structural patterns Structural domains in the polypeptide troponin C

63. Stable folding patterns in proteins Burial of hydrophobic amino acid R groups so as to exclude water requires at least two layers of secondary structures.

64. Stable folding patterns in proteins Connections between elements of secondary structure cannot cross or form knots.

65. Stable folding patterns in proteins Two parallel b strands must be connected by a crossover strand. Right-handed connections tend to be shorter and bend through smaller angles, making them easier to form.

66. Stable folding patterns in proteins

67. Constructing large motifs from smaller ones

68. 6. Protein motifs are the basis for protein structural classification SCOP databases in PDB.

69. 6. Protein motifs are the basis for protein structural classification

72. 7. Protein quaternary structures range from simple dimers to large complexes Quaternary structure of deoxyhemoglobin

73. Rotational symmetry in proteins

76. Viral capsids

77. 1.Overview of Protein Structure 2.Protein Secondary Structure 3.Protein Tertiary and Quaternary Structures 4.Protein Denaturation and Folding Three-Dimensional Structure of Proteins

78. 1. Loss of protein structure results in loss of function. 2. Amino acid sequence determines tertiary structure. 3. Polypeptides fold rapidly by a stepwise process. 4. Some proteins undergo assisted folding. Protein Denaturation and Folding

79. 1. Loss of protein function results in loss of function

80. Circular Dichrosim (CD) Spectroscopy - Introduction 1. When plane polarized light passes through a solution containing an optically active substance the left and right circularly polarized components of the plane polarized light are absorbed by different amounts. 2. When these components are recombined they appear as elliptically polarized light. The ellipticity is defined as . 3. CD is the ellipticity (difference) in absorption between left and right handed circularly polarized light that measured with spectropolarimeter. 4. Proteins and nucleic acids contain elements of asymmetry and thus exhibit distinct CD signals.

81. Far-UV (180-250 nm) CD for determining protein secondary structure Secondary Structure Signal (+/-) WL (nm)  -helix +190-195 -208 -222  -sheet +195-200 -215-220 random-200 coil+220

82. Near-UV (250-350 nm) CD is dominated by aromatic amino acids and disulfide bonds a.a. residueAbs max. (nm) Phe 254, 256 262, 267 Tyr 276 Trp282 Disulfides250-300 broad band

83. Circular Dichrosim (CD) Spectroscopy - Applications 1. Secondary structure content of macromolecules 2. Conformation of proteins and nucleic acids - Effects of salt, pH, and organic solvents 3. Kinetics - Protein folding, unfolding, denaturation or aggregation 4. Thermodynamics - Protein stability to temperature or chemical denaturants

84. Circular Dichrosim (CD) Spectroscopy

85. 2. Amino acid sequence determines tertiary structure

86. 3. Polypeptide fold rapidly by a stepwise process

87. The thermodynamics of protein folding depicted as a free-energy funnel

88. Death by misfolding: the prion diseases 1. A misfolded protein appears to be the causative agent of a number of rare degenerative brain diseases in mammals. 2. Mad cow disease (bovine spongiform encephalopathy, BSE) 3. Related diseases: Human- kuru, Creutzfeldt-Jakob disease (CJD) Sheep- scrapie Deer and Elk- chronic wasting disease 4. Typical symptoms: dementia and loss of coordination, fatal

89. Death by misfolding: the prion diseases A stained section of the cerebral cortex from a patient with Creutzfeld-Jakob disease shows spongiform degeneration.

90. Death by misfolding: the prion diseases 1. Prusiner S. provided evidence that the infectious agent has been traced to a single protein (M r 28,000), prion (PrP). 2. Role of PrP: molecular signaling function in brain tissues 3. Strains of mice lacking the gene for PrP suffer no ill effects. 4. Illness occurs when the normal cellular PrP c occurs in an altered conformation called PrP Sc. 4. Interaction of PrP Sc with PrP c converts the latter to PrP Sc, initiating a domino effect in which more and more of the normal cellular protein converts to the disease-causing form.

91. Death by misfolding: the prion diseases The structure of human PrP in monomeric and dimeric forms.

92. 4. Some proteins undergo assisted folding Folding Accessory Proteins

93. 4. Some proteins undergo assisted folding Protein Disulfide Isomerase (PDI)

94. 4. Some proteins undergo assisted folding Peptidyl Prolyl Isomerase (PPI)

95. 4. Some proteins undergo assisted folding Molecular chaperones

96. 4. Some proteins undergo assisted folding Unrelated classes of chaperones

97. 4. Some proteins undergo assisted folding- Chaperones

98. 4. Some proteins undergo assisted folding- Chaperonins Chaperonin: GroEL/GroES

99. 4. Some proteins undergo assisted folding- Chaperonins