FMOC Chemical Synthesis - ANSWER: Used in synthesis of a growing amino acid
chain to a polystyrene bead. FMOC is used as a protecting group on the N-terminus.
Salting Out (Purification) - ANSWER: Changes soluble protein to solid precipitate.
Protein precipitates when the charges on the protein match the charges in the
solution.
Size-Exclusion Chromatography - ANSWER: Separates sample based on size with
smaller molecules eluting later.
Ion-Exchange Chromatography - ANSWER: Separates sample based on charge. CM
attracts +, DEAE attracts -. May have repulsion effect on like charges. Salt or acid
used to remove stuck proteins.
Hydrophobic/Reverse Phase Chromatography - ANSWER: Beads are coated with a
carbon chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent
(acetonitrile).
Affinity Chromatography - ANSWER: Attach a ligand that binds a protein to a bead.
Elute with harsh chemicals or similar ligand.
SDS-PAGE - ANSWER: Uses SDS. Gel is made from cross-linked polyacrylamide.
Separates based off of mass with smaller molecules moving faster. Visualized with
Coomassie blue.
SDS - ANSWER: Sodium dodecyl sulfate. Unfolds proteins and gives them uniform
negative charge.
Isoelectric Focusing - ANSWER: Variation of gel electrophoresis where protein charge
matters. Involves electrodes and pH gradient. Protein stops at their pI when neutral.
FDNB (1-fluoro-2,3-dinitrobenzene) - ANSWER: 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.
,Iodoacetate - ANSWER: Adds carboxymethyl group on free -SH groups. Blocks
disulfide bonding.
Homologs - ANSWER: Shares 25% identity with another gene
Orthologs - ANSWER: Similar genes in different organisms
Paralogs - ANSWER: Similar "paired" genes in the same organism
Ramachandran Plot - ANSWER: Shows favorable phi-psi angle combinations. 3 main
"wells" for α-helices, ß-sheets, and left-handed α-helices.
Glycine Ramachandran Plot - ANSWER: Glycine can adopt more angles. (H's for R-
group).
Proline Ramachandran Plot - ANSWER: Proline adopts fewer angles. Amino group is
incorporated into a ring.
α-helices - ANSWER: 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.
Helix Dipole - ANSWER: Formed from added dipole moments of all hydrogen bonds
in an α-helix. N-terminus is δ+ and C-terminus is δ-.
ß-sheet - ANSWER: Either parallel or anti-parallel. Often twisted to increase strength.
Parallel ß-sheet - ANSWER: Same sheet directions (C & N-termini line up). Has angled
H-bonds.
ß-turns - ANSWER: Tight u-turns with specific phi-psi angles. Must have gly at
position 3. Proline may also be at ß-turn because it can have a cis-omega angle.
Loops - ANSWER: Not highly structured. Not necessary highly flexible, but can
occasionally move. Very variable in sequence.
Circular Dichroism - ANSWER: Uses UV light to measure 2° structure. Can be used to
measure destabilization.
Disulfide-bonds - ANSWER: Bonds between two -SH groups that form between 2°
and 3° structure.
α/ß Protein Folding - ANSWER: Less distinct areas of α and ß folding.
α+ß Protein Folding - ANSWER: Two distinct areas of α and ß folding.
Mechanism of Denaturants - ANSWER: Highly soluble, H-binding molecules. Stabilize
protein backbone in water. Allows denatured state to be stabilized.
Temperature Denaturation of Protein - ANSWER: Midpoint of reaction is Tm.
Cooperative Protein Folding - ANSWER: Folding transition is sharp. More reversible.
Folding Funnel - ANSWER: Shows 3D version of 2D energy states. Lowest energy is
stable protein. Rough funnel is less cooperative.
Protein-Protein Interfaces - ANSWER: "Core" and "fringe" of the interfaces. Core is
more hydrophobic and is on the inside when interfaced. Fringe is more hydrophilic.
π-π Ring Stacking - ANSWER: Weird interaction where aromatic rings stack on each
other in positive interaction.
σ-hole - ANSWER: Methyl group has area of diminished electron density in center;
attracts electronegative groups
Fe Binding of O2 - ANSWER: Fe2+ binds to O2 reversible. Fe3+ has an additional +
charge and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments.
Kd for binding - ANSWER: Kd = [L] when 50% bound to protein.
Kd = 1/Ka
High-Spin Fe - ANSWER: Electrons are "spread out" and result in larger atom.
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