1. Updates and reminders Carbohydrate lab reports due Mon., March 31
Lab check-out this week, return to your assigned lockers
Midterm results
Avg 70
High 97
Low 23
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2. Biochemistry I L26- 26 Mar. 2007 11/15/2018 S.A. McFarland©2008
This is green fluorescent protein (GFP), one of the most widely used proteins in biochemical research. The picture illustrates the principle of GFP in that the green fluorescent emission is induced by blue light excitation. The chromophore (Ser-Tyr-Gly)is represented by spacefill atoms while the beta barrel is represented by thin ribbons.
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3. From amino acids to proteins
Four levels of protein structure
Primary structure- sequence
Secondary structure- local folding
Tertiary structure- overall folding
Quaternary structure- multichain association
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4. The α-Helix Pro and Gly destablize the helix
Amino acid sequence affects stability
Pro and Gly destablize the helix
Gly too floppy
Pro too rigid
Negatively charged residues near the C-terminus or positively charged residues near the N-terminus destabilize the helix
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5. Stereo view of right-handed a-helix
Side chains point outward from the helix axis
hydrophilic residues
hydrophobic residues
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6. Helix in horse liver alcohol dehydrogenase
Hydrophilic residues cluster on one side of the helical wheel
Amino acid sequence
Helical wheel diagram
B/c 3.6 residues/turn, residues are plotted every 100o along the spiral.
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7. Helix in horse liver alcohol dehydrogenase
Amphipathic a helix (blue ribbon)
Hydrophobic residues (blue) directed inward, hydrophilic (red) outward
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8. The α-Helix
Five constraints affect stability of the helix
Electrostatic repulsion between successive amino acid residues with charged R groups
Bulkiness of adjacent R groups
Interactions between R groups spaced three (or four) residues apart
Occurrence of Pro and Gly residues
Interaction between charged amino acids near the termini and the electric dipole inherent to the helix
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9. β Strands and β sheets
Common type of secondary structure
β strands - polypeptide chains that are almost fully extended
β sheets - multiple β strands arranged side-by-side
β strands are stabilized by hydrogen bonds between C=O and -NH on adjacent strands
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10. Parallel and antiparallel β-strands
Antiparallel arrangement more stable
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11. Stereo view of antiparallel β sheet
Side chains (front β strand) alternate sides
β-Strands twist in a right-handed direction
From influenza virus A neuraminidase
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12. Structure of PHL P2 protein
Blue/purple antiparallel β-sheets within a protein
Stereo view of the β sandwich. Polar residues (red), hydrophobic residues (blue)
From Timothy grass pollen
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13. Secondary structure β-sheet α-helix β-strand rise = 0.15 nm
Note: local folding stabilized by H-bonding between amide hydrogens and carbonyl oxygens of polypeptide backbone.
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14. Loops and turns Type of nonrepetitive secondary structure
Connect a helices and β strands and allow a peptide chain to fold back on itself to make a compact structure
Often contain hydrophilic residues and are found on protein surfaces
Exposed to solvent
H-bond with water
Bend-producing residues include: Pro, Thr, Ser, and Gly
Turns - loops containing 5 residues or less that cause abrupt changes in direction
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15. Reverse turns
Classes based on the phi and psi angles of the residues at positions 2 and 3
The essential difference between them is the orientation of the peptide bond between residues at 2 and 3
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16. Combinations of secondary structure
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17. Domains Pyruvate kinase
Independently folded, compact units in proteins
Domain size: ~25 to ~300 amino acid residues
Domains are connected to each other by loops, bound by weak interactions between side chains
Domains illustrate the evolutionary conservation of protein structure
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18. Structural conservation
Cytochrome c
(a) Tuna (+ heme) (b) Tuna (c) Rice (d) Yeast (e) Bacteria
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19. Folds Green fluorescent protein (GFP)
Domains can be classified by characteristic “folds”
A “fold” is a combination of secondary structures that form the core of a domain (i.e., β-barrel of GFP)
Some domains have simple folds, others have more complex folds
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20. Common domain folds
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21. Higher levels of protein structure
Two major categories
Globular
Polypeptide chains folded into spherical of globular shape
Often contain several types of secondary structure
Make up structures that function as enzymes or regulatory proteins
Fibrous
Polypeptide chains arranged in long strands or sheets
Usually consist largely of a single type of secondary structure
Make up structures that provide support, shape, and protection
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22. Fibrous proteins Adapted for a structural function
Share properties that give strength and/or flexibility to the structures in which they occur
In each case, fundamental structural unit is a simple repeating element of secondary structure
Insoluble in water due to a high concentration of hydrophobic residues both in the interior of the protein and on its surface
Hydrophobic surfaces largely buried by packing many similar polypeptide chains together to form elaborate supramolecular complexes
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23. Structure of hair α-Keratins have evolved for strength
Found in mammals
Constitute most of the dry weight of hair, wool, nails, claws, quills, horns, hooves, and much of the outer layer of skin
α-Keratin helix is a right-handed α helix
Two α helices (parallel) wrap around each other to form a supertwisted, left-handed coiled coil
Supertwisting amplifies the strength of overall structure (like a strong rope)
Helical path of the supertwists is left-handed, where surfaces that touch are made of of hydrophobic residues (R-groups meshed in a regular interlocking pattern)
Rich in hydrophobic residues: Ala, Val, Leu, Ile, Met, and Phe
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24. Structure of hair α-Keratins exhibit higher order quaternary structure
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25. Structure of hair α-Keratin quaternary structure can be quite complex
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26. α-Keratin Strength can be enhanced through covalent cross-linking
Cross-links are between polypeptide chains within the multihelical “ropes” and between adjacent chains in a supramolecular assembly
In α-keratins, the cross-links are disulfide bonds
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27. α-Keratin
Strength can be enhanced through covalent cross-linking
In the hardest and toughest α-keratins, up to 18% of the residues are cysteines involved in disulfide bonds
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28. Curly vs. straight hair
Determined by relative orientations of disulfide bridges
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29. α-Keratin Permanent waving is biochemical engineering!
α-Helices can be stretched out until they arrive at a fully-extended β conformation when hair is exposed to moist heat; upon cooling they revert to the α-helical conformation
Permanent waving is achieved by adding a solution of a reducing agent (stinky thiol) with heat after the hair is bent around a roller with the appropriate shape
Reducing agent breaks all disulfide bonds; moist heat breaks H-bonds and causes uncoiling of α-helical structure
Reducing solution removed; then an oxidizing agent is added to form new disulfide bonds between Cys residues of adjacent polypeptide chains (not same pairs as before the treatment)
After washing and coiling, new disulfide linkages exert some torsion or twist on the bundles of α-helical coils in the hair fibers
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30. α-Keratin Permanent waving is biochemical engineering! 11/15/2018
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31. α-Keratin Permanent waving is biochemical engineering! 11/15/2018
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32. Collagen Evolved to provide strength
Accounts for 25-35% of total protein in mammals
Most abundant single protein in most vertebrates
Found in connective tissue
Tendons (ropelike fibers that attach muscle to bone)
Cartilage (between bone, permits smooth movement of joints)
Skin (loosely woven fibers)
Bone (forms matrix material on which minerals precipitate)
Cornea
Consists of three left-handed helical chains coiled around each other in a right-handed supercoil (triple helix, about 1000 residues long)
Three amino acids per turn (collagen is more extended than an a helix)
Typically ~35% Gly, 11% Ala, and 21% Pro and 4-Hyp
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33. Collagen triple helix Evolved to provide strength
Repeating tripeptide sequence Gly-X-Pro or Gly-X-4Hyp
Center of three-stranded superhelix (d) not hollow as it appears
Gly is required at tight junction where three chains are in contact
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34. Collagen triple helix
Evolved to provide strength
Multiple repeats of -Gly-X-Y- where X is often proline and Y is often 4-hydroxyproline
Prolines allow sharp twisting of the collagen helix
Every 3rd residue is Gly, located along central axis of a triple helix (other residues cannot fit)
For each -Gly-X-Y- triplet, one interchain H-bond forms between amide H of Gly in one chain and -C=O of residue X in an adjacent chain
No intrachain H-bonds exist in the collagen helix
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35. Collagen triple helix Interchain H-bonding in collagen 11/15/2018
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36. Modified amino acids in collagen
4-Hydroxyproline (can be 3-)
Widespread
H-bonds seem to participate in stabilizing the structure
5-Hydroxylysine
Less frequent
Attachment sites for polysaccharides
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37. Modified amino acids in collagen
Formed by enzyme hydroxylation reactions (require vitamin C) after incorporation of Pro into collagen
Vitamin C deficiency (scurvy)
Failure to hydroxylate Pro
Defective triple helix (skin lesions, fragile blood vessels, bleeding gums)
Unlike most mammals, humans cannot synthesize vitamin C
Ascorbic acid (vitamin C)
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38. Scurvy
Vitamin E deficiency
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39. Collagen fibrils
Variety of associations to form varying degrees of tensile strength
Supramolecular assemblies consisting of triple-helical collagen molecules (sometimes called tropocollagen molecules)
Banded appearance
Each molecule ~300 nm-long
Molecules overlap neighbours by ~64 nm
Remarkable strength (greater tensile strength than steel wire of equal cross-section)
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40. Collagen fibrils Covalent cross-linking for strength
Interhelix (between two tropocollagens)
Schiff base formation
Allysine + lysine
Intrahelix (within a tropocollagen)
Aldol condensation
Allysine + allysine
Tropocollagen-refers to the triple helix of collagen.
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41. Collagen fibrils Inter-helix Schiff-base formation 11/15/2018
Note: nitrogen replaces oxygen and water leaves.
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42. Collagen fibrils Intra-helix Schiff-base formation
Enymatic formation of allysine
Aldol reaction to generate an α,β-unsaturated aldehyde
1,4-Michael addition of N-His
Imine formation between aldol-His and 5-hydroxylysine
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46. Collagen fibrils
Schiff-base formation
Increasingly rigid and brittle character of aging connective tissue results from accumulated covalent cross-links in collagen fibrils
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47. Genetic defects in collagen structure
Osteogenesis imperfecta
Abnormal bone formation in babies, which can be fatal
Substitution of a Ser or Cys for a single Gly residue in each α chain
Single-residue substitution has a catastrophic effect on collagen function because they disrupt the Gly-X-Y repeat that gives collagen its unique helical structure
Gly cannot be replaced by another amino acid residue without substantial deleterious effects on collagen structure
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48. Silk fibroin Protein of silk, produced by insects and spiders
Polypeptide chains are predominantly in the β conformation
Rich in Ala and Gly residues, permitting a close-packing of β sheets and an interlocking arrangement of R groups
Overall structure is stabilized by extensive H-bonding
Silk does not stretch because the β conformation is already highly extended
Structure is flexible because the sheets are held together by numerous weak interactions (rather than by covalent bonds)
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49. Silk fibroin Protein of silk, produced by insects and spiders
Strands of fibroin (blue) emerge from the spinnerets of a spider in this colorized electron micrograph
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50. Globular proteins
Combinations of several types of secondary structure
Tertiary structure results from the folding of a polypeptide chain into a closely-packed three-dimensional structure
Amino acids far apart in the primary structure may be brought together
Stabilized primarily hydrophobic effects determined by side chains (contrast with 2o stabilization)
Disulfide bridges also stabilize tertiary structure after protein has folded
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51. Quiz 05
Draw Asp-Tyr at pH (Assume pKa values of 4.1 for the β-carboxyl group of aspartic acid, 2.2 for a-carboxylic acids, and 9.4 for a-ammonium groups.)
To extract Asp-Tyr into ether, what pH must an aqueous solution containing the dipeptide be?
To extract the DNP-derivative of Asp-Tyr into ether, what pH must an aqueous solution containing the dipeptide be?
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