Used in synthesis of a growing amino acid chain to a polystyrene bead. FMOC is used as a protecting group on the N-terminus.
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Salting Out (Purification)
Changes soluble protein to solid precipitate. Protein precipitates when the charges on the protein match the charges in the solution.
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Size-Exclusion Chromatography
Separates sample based on size with smaller molecules eluting later.
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Ion-Exchange Chromatography
Separates sample based on charge. CM attracts +, DEAE attracts -. May have repulsion effect on like charges. Salt or acid used to remove stuck proteins.
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Hydrophobic/Reverse Phase Chromatography
Beads are coated with a carbon chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent (acetonitrile).
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Affinity Chromatography
Attach a ligand that binds a protein to a bead. Elute with harsh chemicals or similar ligand.
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SDS-PAGE
Uses SDS. Gel is made from cross-linked polyacrylamide. Separates based off of mass with smaller molecules moving faster. Visualized with Coomassie blue.
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SDS
Sodium dodecyl sulfate. Unfolds proteins and gives them uniform negative charge.
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Isoelectric Focusing
Variation of gel electrophoresis where protein charge matters. Involves electrodes and pH gradient. Protein stops at their pI when neutral.
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FDNB (1-fluoro-2,3-dinitrobenzene)
FDNB reacts with the N-terminus of the protein to produce a 2,4-dinitrophenol derivative that labels the first residue. Can repeat hydrolysis to determine sequential amino acids.
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DTT (dithiothreitol)
Reduces disulfide bonds.
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Iodoacetate
Adds carboxymethyl group on free -SH groups. Blocks disulfide bonding.
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Homologs
Shares 25% identity with another gene
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Orthologs
Similar genes in different organisms
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Paralogs
Similar "paired" genes in the same organism
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Ramachandran Plot
Shows favorable phi-psi angle combinations. 3 main "wells" for α-helices, ß-sheets, and left-handed α-helices.
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Glycine Ramachandran Plot
Glycine can adopt more angles. (H's for R-group).
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Proline Ramachandran Plot
Proline adopts fewer angles. Amino group is incorporated into a ring.
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α-helices
Ala is common, Gly & Pro are not very common. Side-chain interactions every 3 or 4 residues. Turns once every 3.6 residues. Distance between backbones is 5.4Å.
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Helix Dipole
Formed from added dipole moments of all hydrogen bonds in an α-helix. N-terminus is δ+ and C-terminus is δ-.
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ß-sheet
Either parallel or anti-parallel. Often twisted to increase strength.
Highly soluble, H-binding molecules. Stabilize protein backbone in water. Allows denatured state to be stabilized.
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Temperature Denaturation of Protein
Midpoint of reaction is Tm.
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Cooperative Protein Folding
Folding transition is sharp. More reversible.
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Folding Funnel
Shows 3D version of 2D energy states. Lowest energy is stable protein. Rough funnel is less cooperative.
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Protein-Protein Interfaces
"Core" and "fringe" of the interfaces. Core is more hydrophobic and is on the inside when interfaced. Fringe is more hydrophilic.
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π-π Ring Stacking
Weird interaction where aromatic rings stack on each other in positive interaction.
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σ-hole
Methyl group has area of diminished electron density in center; attracts electronegative groups
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Fe Binding of O2
Fe2+ binds to O2 reversible. Fe3+ has an additional + charge and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments.
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Ka for Binding
Ka = [PL] / [P][L]
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ϴ-value in Binding
ϴ = (bound / total)x100% ϴ = [L] / ([L] + 1/Ka)
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Kd for binding
Kd = [L] when 50% bound to protein. Kd = 1/Ka
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High-Spin Fe
Electrons are "spread out" and result in larger atom.
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Low-Spin Fe
Electrons are less "spread out" and are compacted by electron rich porphyrin ring.
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T-State
Heme is in high-spin state. H2O is bound to heme.
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R-State
Heme is in low-spin state. O2 is bound to heme.
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O2 Binding Event
O2 binds to T-state and changes the heme to R-state. Causes a 0.4Å movement of the iron.
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Hemoglobin Binding Curve
4 subunits present in hemoglobin that can be either T or R -state. Cooperative binding leads to a sigmoidal curve.
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Binding Cooperativity
When one subunit of hemoglobin changes from T to R-state the other sites are more likely to change to R-state as well. Leads to sigmoidal graph.
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Homotropic Regulation of Binding
Where a regulatory molecule is also the enzyme's substrate.
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Heterotropic Regulation of Binding
Where an allosteric regulator is present that is not the enzyme's substrate.
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Hill Plot
Turns sigmoid into straight lines. Slope = n (# of binding sites). Allows measurement of binding sites that are cooperative.
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pH and Binding Affinity (Bohr Affect)
As [H+] increases, Histidine group in hemoglobin becomes more protonated and protein shifts to T-state. O2 binding affinity decreases.
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CO2 binding in Hemoglobin
Forms carbonic acid that shifts hemoglobin to T-state. O2 binding affinity decreases. Used in the peripheral tissues.
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BPG (2,3-bisphosphoglycerate)
Greatly reduces hemoglobin's affinity for O2 by binding allosterically. Stabilizes T-state. Transfer of O2 can improve because increased delivery in tissues can outweigh decreased binding in the lungs.