Protein Structure Peptide Bond

I. Protein Structure A. Primary 1. Peptide Bond Planar, strong, f/y angles a. 2. Determination a. Sequence determination; CHEMICAL i. aa composition; Divide & conquer; Edman degradation b. Sequence determination; PHYSICAL i. Tandem Mass Spectrometry for proteins c. Sequence determination; BIOLOGICAL i. Genome sequenced; need partial sequence d. Determination of Disulfide bonds B. Secondary 1. 2. Conformational structure; Levinthal paradox Pauling & Corey’s predictions a-Helix b-sheets/strands Connections between b-strands Connections between a-helices; angle not important a. b. c. d. 3. Super secondary structure 1. 2. 3. 4. 5. Picturing and classifications Topology Domains Intrinsically disordered Stability 1. 2. Nomenclature Stability C. Tertiary D. II. Quaternary Protein Structure Determination A. Quaternary Protein Structure-Tertiary Protein Classification: α, β, α/β, or barrels Ribbon Topology Greek Key Immunoglobulin-fold Cytochrome b562 PDBid 256B Human immunoglobulin fragment PDBid 7FAB Rossmann fold Rossmann fold Dogfish lactate dehydrogenase domain 2 PDBid 6LDH 1 Protein Structure-Tertiary Protein Classification: α/β Flavodoxin Protein Structure-Tertiary Protein Classification: 8-Stranded Barrels (either β or α/β) b-meander Greek key a/b-barrel Human retinol binding protein Triose phosphate isomerase PDBid 1RBP PDBid 1TIM Peptide-N4-(N-acetyl-b-D-glucosaminyl) asparagine amidase PDBid 1PNG 2 Protein Structure-Tertiary Protein Domains Domains: Separate folded structures with hinge all with one polypeptide chain Recall: g-B Crystallin Hinge region 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 3 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) 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 4 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 5 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 6 Protein Structure-Tertiary Hydrophobic effect drives protein folding Hydropathy Plot Protein Structure-Tertiary Globular proteins • a hydrophobic core and a hydrophilic surface 42 7 Quaternary Structure Protein Structure-Quaternary All subunits colored differently a3 homo trimer Subunits with different sequences colored differently 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 8 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 9 Protein Structure-Summary 47 Protein-Conformational Structure Determination Quaternary Structure 10 Protein-Conformational Structure Determination Determination of Quaternary Structure Need the “native” molecular weight Gel filtration Ultracentrifugation Need the subunit stoichiometry and molecular weight MALDI SDS-PAGE MALDI-MS Example 1: Example 3: Example 2: Native MW = 200 kDa Native MW = 360 kDa Native MW = 280 kDa Subunit MW = 50 kDa Subunit MW = 80 kDa, 40 kDa Subunit MW = 80 kDa, 40 kDa Subunit stoichiometry = equal Subunit stoichiometry = (means with half the size, the smaller one would (80:40) 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 11 Protein-Conformational Structure Determination Quaternary Structure 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 12 Protein-Conformational Structure Determination Analytical Ultracentrifugation In analytical ultracentrifugation, analytes are separated on the basis of their sedimentation when they experience a centrifugal force ~1.4 g/cm3 (Separation is based on Mass, Shape, Density). Usually carried out at speeds around 60,000 rpm There is a force due to buoyancy Fb and a frictional force on the molecule Ff, (centripetal force) both which oppose its movement through the medium Fs (centrifugal or sedimentary force). Protein-Conformational Structure Determination TWO Types: Analytical Ultracentrifugation – Sedimentation Velocity Analytical Ultracentrifugation – Sedimentation Equilibrium Or, described mathematically…Fs + Fb + Ff = Sed. Vel. (rate) Solvent density in g/mL Specific Volume (occupied by 1 g of solute (cm3/g)) M is number average molecular weight (mN). If all species are the same, then M = Mr. Units are g/mol. sedimentation velocity angular velocity M (1 -n r ) µs = 2 ºs Nf wr radius from center of spin Sedimentation coefficient (units of sec) coefficient of friction (g/mol•sec) 13 Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Velocity M (1 -n r ) µs = 2 ºs Nf wr Units of sedimentation are seconds: Sedimentation coefficient (units of sec) 1 Svedberg = 1S = 10-13 s 1 -n r is called the buoyancy factor – and since 1/n approximates r, then if the (Say, “Vee-bar-rho”) density of the molecule is close to that of the solution 1 -n r = 0 and the s = 0. Proteins are more dense than solution and 1 -n r 1 -n r < 0 and the molecule floats. > 0, and the molecule sediments. !⩟ If the molecule is less dense than solution then (lipids or fats in a blood sample) 14