ACS BIOCHEMISTRY EXAM QUESTIONS AND ANSWERS

ACS BIOCHEMISTRY EXAM QUESTIONS AND ANSWERS Henderson-Hasselbach Equation - answerpH = pKa + log ([A-] / [HA]) FMOC Chemical Synthesis - answerUsed 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) - answerChanges soluble protein to solid precipitate. Protein precipitates when the charges on the protein match the charges in the solution. Size-Exclusion Chromatography - answerSeparates sample based on size with smaller molecules eluting later. Ion-Exchange Chromatography - answerSeparates 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 - answerBeads are coated with a carbon chain. Hydrophobic proteins stick better. Elute with non-H-bonding solvent (acetonitrile). Affinity Chromatography - answerAttach a ligand that binds a protein to a bead. Elute with harsh chemicals or similar ligand. SDS-PAGE - answerUses SDS. Gel is made from cross-linked polyacrylamide. Separates based off of mass with smaller molecules moving faster. Visualized with Coomassie blue. SDS - answerSodium dodecyl sulfate. Unfolds proteins and gives them uniform negative charge. Isoelectric Focusing - answerVariation 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) - answerFDNB 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. DTT (dithiothreitol) - answerReduces disulfide bonds. Iodoacetate - answerAdds carboxymethyl group on free -SH groups. Blocks disulfide bonding. Homologs - answerShares 25% identity with another gene Orthologs - answerSimilar genes in different organisms Paralogs - answerSimilar "paired" genes in the same organism Ramachandran Plot - answerShows favorable phi-psi angle combinations. 3 main "wells" for α-helices, ß-sheets, and left-handed α-helices. Glycine Ramachandran Plot - answerGlycine can adopt more angles. (H's for R-group). Proline Ramachandran Plot - answerProline adopts fewer angles. Amino group is incorporated into a ring. α-helices - answerAla 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 - answerFormed from added dipole moments of all hydrogen bonds in an α- helix. N-terminus is δ+ and C-terminus is δ-. ß-sheet - answerEither parallel or anti-parallel. Often twisted to increase strength. Anti-parallel ß-sheet - answerAlternating sheet directions (C & N-termini don't line-up). Has straight H-bonds. Parallel ß-sheet - answerSame sheet directions (C & N-termini line up). Has angled H-bonds. ß-turns - answerTight 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 - answerNot highly structured. Not necessary highly flexible, but can occasionally move. Very variable in sequence. Circular Dichroism - answerUses UV light to measure 2° structure. Can be used to measure destabilization. Disulfide-bonds - answerBonds between two -SH groups that form between 2° and 3° structure. ß-mercaptoethanol - answerBreaks disulfide bonds. α-keratin - answerformed from 2 α-helices twisted around each other. "Coiled coil". Cross- linked by disulfide bonds. Collagen - answerRepeating sequence of Gly-X-Pro. 3 stranded "coiled coil". Contains gly core. Myoglobin 4° Structure - answerSymmetric homodimer, Hemoglobin 4° Structure - answerTetramer. Dimer of dimers. α2ß2 tetramer. α/ß Protein Folding - answerLess distinct areas of α and ß folding. α+ß Protein Folding - answerTwo distinct areas of α and ß folding. Mechanism of Denaturants - answerHighly soluble, H-binding molecules. Stabilize protein backbone in water. Allows denatured state to be stabilized. Temperature Denaturation of Protein - answerMidpoint of reaction is Tm. Cooperative Protein Folding - answerFolding transition is sharp. More reversible. Folding Funnel - answerShows 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 - answerWeird interaction where aromatic rings stack on each other in positive interaction. σ-hole - answerMethyl group has area of diminished electron density in center; attracts electronegative groups Fe Binding of O2 - answerFe2+ binds to O2 reversible. Fe3+ has an additional + charge and binds to O2 irreversibly. Fe3+ rusts in O2 rich environments. Ka for Binding - answerKa = [PL] / [P][L] ϴ-value in Binding - answerϴ = (bound / total)x100% ϴ = [L] / ([L] + 1/Ka) Kd for binding - answerKd = [L] when 50% bound to protein. Kd = 1/Ka High-Spin Fe - answerElectrons are "spread out" and result in larger atom. Low-Spin Fe - answerElectrons are less "spread out" and are compacted by electron rich porphyrin ring. T-State - answerHeme is in high-spin state. H2O is bound to heme. R-State - answerHeme is in low-spin state. O2 is b