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Category,Question,Answer

Protein folding & misfolding (exam practice),"The Alzheimer's beta-peptide and prion proteins both convert from a soluble form to an amyloid form. What conformational rearrangement underlies this, and why does it enable amyloid formation?","Both undergo a rearrangement from alpha-helix to beta-strand/beta-sheet structure; amyloid fibrils are built from propagating intermolecular beta-sheet structures, so the presence of beta-strands is required for amyloid to form."

Protein folding & misfolding (exam practice),"What do the Hsp70 and GroEL/GroES chaperone systems have in common, functionally and mechanistically?","Both help fold nascent polypeptides (or refold improperly folded proteins), and both rely on ATP hydrolysis to drive their conformational cycles."

Protein folding & misfolding (exam practice),Cystic fibrosis can be considered a protein-folding disease caused by an amino acid mutation (deltaF508 in CFTR). Is this best described as a gain-of-function or loss-of-function disease?,"Loss of function -- the mutant CFTR protein misfolds and fails to reach/function at the cell membrane, rather than gaining a new toxic activity."

Protein folding & misfolding (exam practice),An Ile residue in a protein's hydrophobic core is mutated to Lys. What's the best explanation for whether this changes protein structure?,"Yes, a structural change is expected -- Lys is positively charged at pH 7 while Ile is uncharged; placing a charged residue in the buried, water-excluded hydrophobic core is highly destabilizing."

Protein folding & misfolding (exam practice),A Lys residue is mutated to a Glu residue elsewhere in a protein. Will this definitely cause a folding change?,"Not necessarily -- it depends on where the Lys is located (e.g., whether it was buried, involved in a salt bridge, or on a fully solvent-exposed surface with no specific role); a single substitution does not automatically disrupt folding."

Protein folding & misfolding (exam practice),What kind of chemical bond do disulfide bonds represent in the context of protein tertiary structure stability?,"A covalent bond -- distinct from the non-covalent interactions (H-bonds, van der Waals, electrostatic, hydrophobic effect) that otherwise stabilize tertiary structure."

Secondary & tertiary structure (exam practice),Can two parallel beta-strands be connected directly by a short beta-turn? Why or why not?,"No -- in a parallel beta-sheet, the N-terminus of one strand lines up with the N-terminus (not C-terminus) of the neighboring strand, so the strand ends that would need to connect are not in close proximity; beta-turns are only 3-4 residues long, too short to span that distance. (Antiparallel strands, whose adjacent ends ARE close together, can be connected by a short turn.)"

Secondary & tertiary structure (exam practice),True or False (trick question): the characteristic repeating sequence of alpha-keratin is (Gly-Pro-Hyp).,False -- (Gly-Pro-Hyp) is collagen's repeating motif. Keratin is a coiled-coil alpha-helical protein with no such simple tripeptide repeat.

Secondary & tertiary structure (exam practice),"Which of the following is NOT a fibrous protein: silk fibroin, keratin, myoglobin, collagen?","Myoglobin -- it is a globular, all-alpha-helical oxygen-storage protein, not a structural fibrous protein."

Secondary & tertiary structure (exam practice),"Which amino acid side chain would most likely be found in the interior of a water-soluble, globular protein: Glu, Asp, Lys, Arg, or Ile?","Ile -- it is hydrophobic and nonpolar, so it is favorably buried in the protein's interior; the other four (Glu, Asp, Lys, Arg) are charged and favor the solvent-exposed surface."

Secondary & tertiary structure (exam practice),Does an alpha-helix have a net macroscopic dipole? Why?,"Yes -- because every backbone amide dipole (C=O toward the C-terminal direction) points the same way along the helix axis, the individual dipoles sum to give the helix an overall macrodipole: partial positive charge near the N-terminus and partial negative charge near the C-terminus."

Secondary & tertiary structure (exam practice),True or False (trick question): the alpha-helix has about seven amino acid residues per helical turn.,False -- an alpha-helix has 3.6 residues per turn (7 is not correct for any standard helix discussed in this course).

Secondary & tertiary structure (exam practice),"For the helix-forming sequence AFHAYSEMASALD (positions 1-13), which two residues are positioned to form a favorable salt bridge at pH 5, and why?","His (position 3) and Glu (position 7) -- they are separated by i, i+4 spacing (one helical turn apart), placing them on the same face of the helix close enough to interact; Asp (position 13) is too far away to make a similar contact."

Secondary & tertiary structure (exam practice),"Roughly what distance (in Angstroms) separates two side chains at i, i+4 spacing within an alpha helix (e.g., a salt-bridge pair one turn apart)?","About 5.4 Angstroms -- this is the helix's pitch (rise per turn), since i, i+4 residues are exactly one full turn apart."

Secondary & tertiary structure (exam practice),"On a Ramachandran plot, how can you tell which of two plots corresponds to a mostly-beta-sheet protein versus a mostly-alpha-helical protein?",The mostly-beta protein's plot will show many points in the upper-left quadrant (the beta-sheet region); the mostly-alpha-helical protein's plot will show points concentrated in the lower-left quadrant (the right-handed alpha-helix region) with few or no points in the beta region.

Secondary & tertiary structure (exam practice),Why don't Ramachandran plots typically show variation in the omega (amide) dihedral angle?,"Because the peptide bond has partial double-bond character (resonance) and is almost always trans (omega ~180 degrees) in natural proteins; since there is very little variation, there's no need to plot it as a separate dimension."

Amino acids & pI (exam practice),Which two standard amino acids have a stereocenter located in their side chain (in addition to the alpha-carbon)?,Threonine and Isoleucine -- both have a beta-carbon bonded to four different substituents (a second chiral center) due to their branched side chains.

Amino acids & pI (exam practice),"At what pH is a Cys residue's thiol side chain most reactive (as a nucleophile), and why?","Around pH 8 (at or above its pKa, ~8) -- above the pKa the thiol is increasingly deprotonated into the thiolate (-S-) form, which is a much stronger nucleophile than the neutral thiol (-SH)."

Amino acids & pI (exam practice),"Using Henderson-Hasselbalch, what percentage of Lysine residues are in the charged (protonated) form at pH 8, given pKa(Lys) ~ 10.5?","About 99.9% -- fraction charged = 1 / (1 + 10^(pH-pKa)) = 1 / (1 + 10^(8-10.5)) = 1 / (1 + 10^-2.5) is approximately 0.999, i.e., ~99.9% still protonated/charged, since pH is far below the pKa."

Amino acids & pI (exam practice),"For the peptide PRIME (Pro-Arg-Ile-Met-Glu), what is the net charge at pH 9 (using pKa's: N-term ~8, Arg ~12.5, Glu ~4.1, C-term ~3.1)?","-1 -- N-terminus is deprotonated/neutral (pH 9 is above its pKa ~8), Arg is protonated (+1), Ile and Met are uncharged, Glu is deprotonated (-1), and the C-terminus is deprotonated (-1); sum = 0 + 1 - 1 - 1 = -1."

Amino acids & pI (exam practice),"For the peptide WINTER, what is its net charge at pH 2 (strongly acidic)?","+2 -- at low pH, the N-terminus and any basic side chain(s) are fully protonated (positive), while acidic groups (C-terminus, any Asp/Glu) remain protonated/neutral, giving a net positive charge overall for this sequence."

Amino acids & pI (exam practice),"If a peptide has a net charge of 0 at the pH you're working at, can you purify it by ion-exchange chromatography?","No -- with zero net charge, the peptide won't bind appreciably to either a cation-exchange (negative) or anion-exchange (positive) resin, so ion-exchange chromatography cannot separate it at that pH."

Amino acids & pI (exam practice),"At pH 10, should a Cys residue in a peptide be drawn as the neutral thiol (-SH) or the deprotonated thiolate (-S-)?","Deprotonated thiolate (-S-) -- pH 10 is well above Cys's pKa (~8), so the thiol should be shown ionized/deprotonated."

Amino acids & pI (exam practice),"Would you expect a molecule like NH3 (ammonia) to hydrogen-bond with water? What about CH4, NaCl, or CaCl2?","Yes for NH3 -- it has both a lone pair (H-bond acceptor) and N-H bonds (H-bond donor). CH4 has no polar bonds or lone pairs available and cannot H-bond; NaCl and CaCl2 are ionic salts that dissociate into ions and interact with water via ion-dipole forces/hydration, not classic hydrogen bonding."

Protein purification (exam practice),"You purify a disulfide-linked homodimeric protein by size-exclusion chromatography, then repeat the run in the presence of beta-mercaptoethanol (BME). Will the protein elute faster, slower, or the same, and why?","Slower (it will take longer to elute) -- BME reduces the disulfide crosslink, converting the dimer into smaller monomers; smaller proteins penetrate further into the porous matrix beads used in size-exclusion chromatography, taking a longer path (and thus more time) through the column than larger proteins, which are excluded from the pores and elute first."

Protein purification (exam practice),"Which type of chromatography would you use to separate proteins based on a specific binding interaction (e.g., to an immobilized ligand or antibody)?",Affinity chromatography.

Protein purification (exam practice),You have two proteins with different thermal stabilities (different melting temperatures). How can you use this (a CHASM property) to purify the more stable one?,"Heat the mixture to a temperature between the two melting points; the less stable protein will denature and precipitate/aggregate out of solution, while the more stable target protein remains folded and soluble. Centrifuge to pellet the precipitated protein, leaving the purified target protein in the supernatant."

Structure determination & analysis tools (exam practice),"Name two protein sequencing/analysis tools mentioned in lecture, besides X-ray, NMR, and cryo-EM.",Edman degradation and mass spectrometry (both used to help determine a protein's amino acid sequence).

Structure determination & analysis tools (exam practice),Why can't mass spectrometry alone always distinguish between two different amino acids in a peptide sequence?,"Because some amino acids share (nearly) the same mass -- for example, Isoleucine and Leucine are isomers with identical molecular formulas/mass, so MS alone cannot tell them apart based on mass."

Amino acids & pI (exam practice),What level of protein structure describes how multiple folded subunits (polypeptide chains) assemble into a complex like hemoglobin?,Quaternary structure.

Amino acids & pI (exam practice),Why do free (unincorporated) amino acids have different terminal pKa's than the same groups once incorporated into a peptide? Give the typical values for each.,"The alpha-carboxyl and alpha-amino groups of a FREE amino acid are both adjacent to the same charged/polar alpha-carbon environment, giving pKa's of about 2 (alpha-COOH) and about 9 (alpha-NH3+). Once in a peptide, only the terminal residues retain a free alpha-amino or alpha-carboxyl group, and the adjacent peptide bond changes the electronic environment, shifting the values to about 3 (C-terminal carboxyl) and about 8 (N-terminal amino)."