lecture 10 (9/30/20) · 2020. 9. 30. · o homo vs. hetero o subunits indicated with greek letters...

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• Reading: Ch4; 125, 138-141, 141-142

• Problems: Ch4 (text); 7, 9, 11Ch4 (study guide); 1, 2

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• 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


α

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Human immunoglobulin fragmentPDBid 7FAB


β

Immunoglobulin-fold


α/β

Flavodoxin

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α/β

Flavodoxin


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


Greek key

(4-5-6-7)

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Triose phosphate isomerasePDBid 1TIM

a/b-barrel


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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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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


Tachystatin

Intrinsically Disordered Proteins: Tachystatin

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25


E3ubiquitin-proteinligaseMdm2

N

N

C

Intrinsically Disordered Proteins: p53

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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


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


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Globularproteins

• a hydrophobic core and a hydrophilic surface

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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



• Saltbridges• vanderWaals(Dispersionforces)

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Sum of weak interactions


• Saltbridges• vanderWaalsforces

4° structure held together with side-chain interactions


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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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

lecture 10 (9/30/20) · 2020. 9. 30. · o homo vs. hetero o subunits indicated with greek letters...

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