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• Reading: Ch4; 125, 138-141, 141-142
• Problems: Ch4 (text); 7, 9, 11Ch4 (study guide); 1, 2
NEXT
• Reading: Ch4; 125 (Fig 4-10), 134-136 (struct. determin.)• Problems: Ch4 (text); 10, 15
Lecture10(9/30/20)
Lecture10(9/30/20)OUTLINEI. Protein Structure
A. Primary1. Determination
a. Sequence determination; CHEMICALb. Sequence determination; PHYSICALc. Sequence determination; BIOLOGICAL
B. Secondary1. Conformational structure; Levinthal paradox2. Pauling & Corey’s predictions
a. a-Helixb. b-sheets/strandsc. Connections between b-strandsd. Connections between a-helices; angle not important
3. Super secondary structure C. Tertiary
1. Picturing and classifications2. Topology3. Domains4. Intrinsically disordered5. Stability
D. QuaternaryII. Protein Characterization; Structure determination
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Tertiary Structure
Cytochrome b562PDBid 256B
Human immunoglobulin fragmentPDBid 7FAB
DogfishlactatedehydrogenasePDBid6LDH
Protein Structure-TertiaryProtein Classification:
α, β, α/β, or barrels
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Protein Structure-Tertiary
Cytochrome b562PDBid 256B
Protein Classification: α
Growth hormone
Protein Structure-TertiaryProtein Classification:
α
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Human immunoglobulin fragmentPDBid 7FAB
Protein Structure-TertiaryProtein Classification:
β
Immunoglobulin-fold
Protein Structure-TertiaryProtein Classification:
α/β
Flavodoxin
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Protein Structure-TertiaryProtein Classification:
α/β
Flavodoxin
Protein Structure-Tertiary
Human retinol binding proteinPDBid 1RBP
Peptide-N4-(N-acetyl-b-D-glucosaminyl) asparagine amidasePDBid 1PNG
Triose phosphate isomerasePDBid 1TIM
Protein Topology: 8-Stranded β Barrels
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Human retinol binding proteinPDBid 1RBP
Protein Structure-TertiaryProtein Topology: 8-Stranded β Barrels
b-meander
Peptide-N4-(N-acetyl-b-D-glucosaminyl)asparagineamidasePDBid1PNG
Protein Structure-TertiaryProtein Topology: 8-Stranded β Barrels
Greek key
(4-5-6-7)
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Protein Structure-TertiaryProtein Topology: 8-Stranded β Barrels
Triose phosphate isomerasePDBid 1TIM
a/b-barrel
Protein Structure-Tertiary
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Protein Topology: 8-Stranded β Barrels
a/b-barrels
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g-BCrystallin
Domains: Separate folded structures with hingeall with one polypeptide chain
Protein Structure-TertiaryProtein Domains
Hingeregion
Recall:
1 23
456
DogfishlactatedehydrogenasePDBid6LDH
Protein Structure-TertiaryProtein Domains
a/b-saddle&
Rossmann Fold (b-a-b-a-b)
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Protein Structure-Tertiary
2-Domain Protein : GAPDH
Glyceraldehyde-3-phosphatedehydrogenasePDBid1GD1
a/b-saddle&
Rossmann Fold (b-a-b-a-b)
Protein Domains
Hingeregion
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Unstructured domains: Part of primary structure without any defined conformation, changeable, dynamic, several possible structures depending on binding partners in cells
Protein Structure-Tertiary
Tachystatin
Intrinsically Disordered Proteins: Tachystatin
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Protein Structure-Tertiary
E3ubiquitin-proteinligaseMdm2
N
N
C
Intrinsically Disordered Proteins: p53
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Protein Structure-Tertiary
E3ubiquitin-proteinligaseMdm2
N
N
C
Intrinsically Disordered Proteins: p53
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Protein Structure-TertiaryIntrinsically Disordered
Proteins: p53
Intrinsically Disordered Proteins: CREB
Kinase-inducibledomainofCBP(CREB-bindingprotein)
PDBid1KDX
Protein Structure-Tertiary
CREB (cAMP response element-binding protein)
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• Hydrophobiceffect• Hydrogenbonds
• Saltbridges• vanderWaals(Dispersionforces)
lysozyme
Protein Structure-Summary Tertiary• 3° structure is a collection of 2° structures in motifs and/or
domains• Sum of weak interactions
• 3° structure held together with side-chain interactions
• Disulfidebonds
Hydrophobic effect drives protein folding
Protein Structure-Tertiary
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Globularproteins
• a hydrophobic core and a hydrophilic surface
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Protein Structure-Tertiary
Quaternary Structure
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• Multiple subunits held together by primarily non-covalent interactions
• Nomenclature: o homo vs. heteroo subunits indicated with Greek letters (a (1st), b (2nd), g (3rd), etc.
a3 a4
a2b2a2 b2 g2
homo trimer homo tetramer
hetero tetramer hetero hexamer
Protein Structure-Quaternary
Sameweakinteractionsdetermine4°structure
Protein Structure-Quaternary
• Hydrophobiceffect• Hydrogenbonds
• Saltbridges• vanderWaals(Dispersionforces)
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Sum of weak interactions
• Hydrophobiceffect• Hydrogenbonds
• Saltbridges• vanderWaalsforces
4° structure held together with side-chain interactions
Protein Structure-Quaternary
Intermolecular salt bridges formed by conserved, charged residues stabilize HY2-Fd interaction. HY2-bound substrate and the iron-sulfur cluster on AtFd2 are shown in stick representation. Important charged residues in AtFd2-HY2 binding interface are viewed from the top of Fdside of the docking complex. Residues identified in this study are shown in stick representation with blue (HY2) and brown (AtFd2) carbon atoms. Red circles indicate the salt bridges in-between two proteins.
Phytochromobilin synthase (HY2)
Ferredoxin (Fd)
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Protein Structure-Summary
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ProblemWhichresiduewouldbemorelikelytobe____?1. Onaglobularprotein’ssurface:Trp orGln2. Intheglobularproteininterior:SerorVal3. Inthemiddleofanalpha-helix:Gly orLeu4. Inabeta-sheet:HisorPro
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Protein Structure-Tertiary
ProteinCharacterization
QuaternaryStructure
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MALDI
Determination of Quaternary Structure
Need the “native” molecular weightGel filtrationUltracentrifugation
Need the subunit stoichiometry and molecular weightSDS-PAGEMALDI-MS
Example2:NativeMW=300kDaSubunitMW=75kDa,50kDaSubunitstoichiometry=
(75:50)is2:3
a2b3
Example1:NativeMW=200kDaSubunitMW=50kDa
a4
Example 3: Native MW = 360 kDaSubunit MW = 80 kDa, 40 kDaSubunit stoichiometry = equal
(means with half the size, the smaller one would have to have half intensity on gel)
a3b3
Protein Characterization: Structure Determination