Protein Structure - Tertiary

I. Protein Structure A. Primary 1. 2. Peptide Bond Determination B. Secondary 1. 2. 3. 4. a-Helix b-sheets/strands Connections between b-strands Super secondary structure C. Tertiary 1. 2. 3. 4. 5. Picturing and classifications Topology Domains Intrinsically disordered Stability D. Quaternary II. Protein-Conformational Structure Determination A. Quaternary structure 1. B. 1. 2. 3. C. How determined; Gel filtration & SDS-PAGE, Ultracentrifugation Tertiary structure X-ray diffraction/crystallography NMR spectroscopy Comparison: NMR versus X-ray crystallography Secondary structure 1. Circular dichroism (CD) Tertiary Structure 1 Protein Structure-Tertiary Protein Classification: α, β, α/β, or barrels Cytochrome b562 PDBid 256B Human immunoglobulin fragment PDBid 7FAB Dogfish lactate dehydrogenase domain 2 PDBid 6LDH Protein Structure-Tertiary Protein Classification: α Cytochrome b562 PDBid 256B Growth hormone 2 Protein Structure-Tertiary Protein Classification: α Protein Structure-Tertiary Protein Classification: β Human immunoglobulin fragment PDBid 7FAB Immunoglobulin-fold 3 Protein Structure-Tertiary Protein Classification: α/β Flavodoxin Protein Structure-Tertiary Protein Classification: α/β Flavodoxin 4 Protein Structure-Tertiary Protein Classification: 8-Stranded Barrels (either β or α/β) Human retinol binding protein Triose phosphate isomerase PDBid 1RBP PDBid 1TIM Peptide-N4-(N-acetyl-b-D-glucosaminyl) asparagine amidase PDBid 1PNG Protein Structure-Tertiary Protein Classification: 8-Stranded Barrels (either β or α/β) Human retinol binding protein PDBid 1RBP b-meander 5 Protein Structure-Tertiary Protein Classification: 8-Stranded Barrels (either β or α/β) Peptide-N4-(N-acetyl-b-D-glucosaminyl) asparagine amidase PDBid 1PNG Greek key (4-5-6-7) Protein Structure-Tertiary Protein Classification: 8-Stranded Barrels (either β or α/β) Triose phosphate isomerase PDBid 1TIM a/b-barrel (Rossmann Folds (b-a-b-a-b)) 6 Protein Structure-Tertiary Protein Classification: 8-Stranded Barrels (either β or α/β) a/b-barrels Protein Structure-Tertiary Protein Domains Domains: Separate folded structures with hinge all with one polypeptide chain Recall: g-B Crystallin Hinge region 7 Protein Structure-Tertiary Protein Domains 6 5 4 1 2 3 Dogfish lactate dehydrogenase domain 2 PDBid 6LDH a/b-saddle & Rossmann Fold (b-a-b-a-b) x 2 Protein Structure-Tertiary Protein Domains 2-Domain Protein : GAPDH Hinge region Glyceraldehyde-3-phosphate dehydrogenase PDBid 1GD1 a/b-saddle & Rossmann Fold (b-a-b-a-b) 8 Protein Structure-Tertiary Intrinsically Disordered Proteins: Tachystatin Unstructured domains: Part of primary structure without any defined conformation, changeable, dynamic, several possible structures depending on binding partners in cells Tachystatin Protein Structure-Tertiary C Intrinsically Disordered Proteins: p53 N N E3 ubiquitin-protein ligase Mdm2 9 Protein Structure-Tertiary C Intrinsically Disordered Proteins: p53 N N E3 ubiquitin-protein ligase Mdm2 Protein Structure-Tertiary Intrinsically Disordered Proteins: p53 PONDR=Predictor of Natural Disordered Regions 10 Protein Structure-Tertiary Intrinsically Disordered Proteins: CREB CREB (cAMP response element-binding protein) Kinase-inducible domain of CBP (CREB-binding protein) PDBid 1KDX 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 lysozyme • Salt bridges • Hydrophobic effect • van der Waals (Dispersion • Hydrogen bonds forces) • Disulfide bonds 11 Protein Structure-Tertiary Hydrophobic effect drives protein folding Hydropathy Plot Protein Structure-Tertiary Globular proteins • a hydrophobic core and a hydrophilic surface 12 Quaternary Structure Protein Structure-Quaternary a3 homo trimer a4 • Multiple subunits held together by primarily noncovalent interactions homo tetramer • Nomenclature: o homo vs. hetero o subunits indicated with Greek letters (a (1st), b (2nd), g (3rd), etc. a2 b2 g2 a2b2 hetero tetramer hetero hexamer (homo dimer of hetero trimers) (abg)2 13 Protein Structure-Quaternary Same weak interactions determine 4° structure • Hydrophobic effect • Hydrogen bonds • Salt bridges • van der Waals (Dispersion forces) Protein Structure-Quaternary 4° structure held together with side-chain interactions Sum of weak interactions Ferredoxin (Fd) Phytochrom obilin synthase (HY2) 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 Fd side 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. • Hydrophobic effect • Hydrogen bonds • Salt bridges • van der Waals forces 14 Protein Structure-Summary Protein-Conformational Structure Determination Quaternary Structure 15 Protein-Conformational Structure Determination Determination of Quaternary Structure Need the “native” molecular weight Gel filtration Ultracentrifugation Need the subunit stoichiometry and molecular weight SDS-PAGE MALDI MALDI-MS Example 1: Example 3: Example 2: Native MW = 200 kDa Native MW = 360 kDa Native MW = 300 kDa Subunit MW = 50 kDa Subunit MW = 75 kDa, 50 kDa Subunit MW = 80 kDa, 40 kDa Subunit stoichiometry = equal Subunit stoichiometry = (means with half the size, the smaller one would (75:50) is 2:3 have to have half intensity on gel) a4 (means with the same amounts of total mass per protein, the smaller one would have to have the same intensity on gel) a2b3 a3b3 Protein-Conformational Structure Determination Example of correlating band intensity with size an stoichiometry: EXAMPLE-4 IgG: a2b2 If a smaller protein has the same intensity as that of a larger protein, it means the smaller one must be there in larger numbers (Example 2). EXAMPLE-5: Darker Native Mr=440 kDa 1x Lighter ~3x (but ~½ the size➔6X) 200 Mr=72, 68, 60, 40 kDa a1b1g1d6 16