Biochem midterm 1

Category,Question,Answer

Non-covalent interactions,What are the five main types of non-covalent interactions covered in Lecture 2?,"Hydrogen bonds, electrostatic/ionic interactions (salt bridges), van der Waals interactions, aromatic (pi-stacking) interactions, and the hydrophobic effect."

Non-covalent interactions,What is a dipole?,A separation of positive and negative charge within a molecule or bond.

Non-covalent interactions,"What is the formula for dipole moment, and what does each term mean?","mu = q x r, where q = magnitude of charge and r = distance between the charges (units: Debye, or Coulomb-meters)."

Non-covalent interactions,What makes a covalent bond polarized?,"One atom (the more electronegative one) draws electron density toward itself, creating partial positive and negative charges (delta+ / delta-)."

Non-covalent interactions,What is the optimal hydrogen bond distance?,2.4 to 2.7 Angstroms.

Non-covalent interactions,"In a hydrogen bond, what is the 'donor'?","The atom bearing the H that is being shared/donated (e.g., the N or O attached to that H); the donor H has a partial positive charge from being attached to an electronegative atom."

Non-covalent interactions,Why does the angle of a hydrogen bond matter?,"H-bond strength is maximal at 180 degrees (linear); as the angle deviates from linear, the H-bond becomes weaker."

Non-covalent interactions,What is the van der Waals radius (r_vdw)?,Half the distance of closest approach between two non-bonded atoms of a given element; atoms interact optimally when the distance between them equals the sum of their van der Waals radii.

Non-covalent interactions,What are the three types of van der Waals interactions?,"(1) dipole-dipole, (2) dipole-induced dipole, (3) induced dipole-induced dipole (London dispersion forces)."

Non-covalent interactions,What causes van der Waals / London dispersion forces?,Transient fluctuations in electron clouds create temporary dipoles that mutually reinforce (induce) dipoles in neighboring atoms.

Non-covalent interactions,What creates 'steric effects' at short interatomic distances?,Electron cloud repulsion when atoms get closer than their combined van der Waals radii; this repulsion constrains 3D biomolecular structure.

Non-covalent interactions,"What is Coulomb's Law, and what does it describe?","V(r) = (1/4*pi*epsilon) x (q1*q2 / r); it describes the potential energy of an electrostatic (ionic) interaction between two charges, where epsilon is the electric permittivity of the medium."

Non-covalent interactions,"What are the relative permittivity (dielectric constant) values for vacuum, water, and a cell membrane/protein interior?",Vacuum: epsilon_r = 1; Water: epsilon_r = 80; Cell membrane/protein interior: epsilon_r = 2 to 4.

Non-covalent interactions,Why are electrostatic (ion-pair) interactions much stronger in a protein's hydrophobic interior than on its surface?,"The interior is nonpolar (low dielectric, similar to vacuum), so charges are not 'shielded'; the surface is exposed to water (high dielectric, epsilon=80), which screens/weakens the electrostatic attraction."

Non-covalent interactions,"Rank hydrogen bonds, van der Waals interactions, and ion pairs by typical strength (strongest to weakest).","Ion pairs (electrostatic) > hydrogen bonds > van der Waals interactions (though ion pairs remain strong at longer range, while van der Waals and H-bonds have a shorter optimal distance)."

Non-covalent interactions,"Per Table 2.1, what is the approximate energy range for a charge-charge (ionic) interaction?",-13 to -17 kJ/mol.

Non-covalent interactions,"Per Table 2.1, what is the approximate energy range for a charge-dipole interaction (H-bond)?",-13 to -21 kJ/mol.

Non-covalent interactions,"Per Table 2.1, what is the approximate energy range for a dipole-dipole interaction (H-bond)?",-2 to -8 kJ/mol.

Non-covalent interactions,"Per Table 2.1, what is the approximate energy range for a van der Waals interaction?",-0.4 to -0.8 kJ/mol.

Non-covalent interactions,What causes aromatic pi-stacking interactions?,"Dispersion (induced dipole-induced dipole) forces; pi-bonds are more polarizable than sigma bonds, producing a stronger induced dipole between stacked aromatic rings."

Non-covalent interactions,"In DNA, roughly how many van der Waals contacts occur in a set of two stacked base pairs?",About 15 van der Waals contacts.

Non-covalent interactions,"Rank P, C, N, H, O, S from lowest to highest electronegativity.","P < H < C < S < N < O (approximately; P and H are close, as are C and S)."

Non-covalent interactions,"Rank these hydrogen bonds from weakest to strongest: S-H...O, N-H...O, O-H...O.",S-H...O (weakest) < N-H...O < O-H...O (strongest) -- strength increases with the electronegativity of the donor atom.

Non-covalent interactions,"Rank cyclohexyl-SH, cyclohexyl-NH2, cyclohexyl-OH, and cyclohexyl-CO2H by increasing H-bond donating ability toward methylamine.",-SH < -NH2 < -OH < -CO2H (weakest to strongest); donor strength increases with the electronegativity/acidity of the donating group.

Non-covalent interactions,"Why is water an excellent solvent for NaCl, while hexane is not, even though neither has a formal charge?","Water's permanent dipole forms ion-dipole hydration shells around Na+ and Cl-, and its high dielectric constant (80) screens/weakens the ionic attraction between them. Hexane is nonpolar (no dipole), cannot hydrate the ions, and has a low dielectric constant, so it cannot stabilize separated ions."

Non-covalent interactions,"Would benzene or cyclohexane show stronger intermolecular van der Waals interactions in pure solution, and why?","Benzene -- its planar, delocalized pi-electron system is more polarizable than cyclohexane's sigma-bonded ring, allowing stronger induced-dipole (dispersion/pi-stacking) interactions between molecules."

Water & hydrophobic effect,What is a clathrate (or 'flickering cluster')?,"An ordered, hydrogen-bonded cage of water molecules that forms around a nonpolar (hydrophobic) solute."

Water & hydrophobic effect,What thermodynamic quantity actually drives the hydrophobic effect?,Entropy -- minimizing the ordered water shell around nonpolar surfaces increases the entropy of the surrounding water.

Water & hydrophobic effect,True or False: the hydrophobic effect is caused by an intrinsic attractive force between nonpolar groups.,"False -- it is driven by the increase in entropy of water that occurs when ordered water molecules around nonpolar surfaces are minimized/released, not by direct attraction between the nonpolar groups themselves."

Water & hydrophobic effect,"When hydrophobic molecules cluster together (e.g., forming a micelle), what happens to the entropy of the system, and why?","Entropy increases, because fewer water molecules are forced into an ordered cage around the (now-reduced) hydrophobic surface area."

Water & hydrophobic effect,"When an enzyme binds a substrate, what happens to the ordered water molecules that previously surrounded both surfaces?","They are displaced/released into the bulk solvent as disordered water, which increases entropy and helps stabilize the binding interaction (along with new H-bonds, ionic, and hydrophobic contacts)."

Water & hydrophobic effect,Approximately what percent of human cell mass is water?,About 70%.

Water & hydrophobic effect,Approximately what percent of human cell mass is protein?,About 15-20%.

Water & hydrophobic effect,What structural feature makes a molecule 'amphipathic'?,It has both a polar/hydrophilic region and a nonpolar/hydrophobic region.

Bioenergetics,What is the Gibbs free energy equation?,delta-G = delta-H - T x delta-S.

Bioenergetics,What does a negative delta-G indicate about a reaction?,It is spontaneous / exergonic.

Bioenergetics,What does a positive delta-G indicate about a reaction?,It is non-spontaneous / endergonic (the reverse reaction is favorable).

Bioenergetics,What does delta-G = 0 indicate?,The system is at equilibrium (reversible).

Bioenergetics,What is the relationship between delta-G-standard-prime and Keq?,delta-G-degree-prime = -RT ln(Keq).

Bioenergetics,"If Keq > 1, what is the sign of delta-G-degree-prime, and which direction is favored?",Negative; formation of products is favored.

Bioenergetics,"If Keq < 1, what is the sign of delta-G-degree-prime, and which direction is favored?",Positive; formation of reactants is favored.

Bioenergetics,What are the 'biochemical standard conditions' used to define delta-G-degree-prime?,"Constant pressure (1 atm), room temperature (298 K), [reactants] = [products] = 1 M initially, pH 7.0, and standard [H2O]."

Bioenergetics,What equation relates the actual delta-G under cellular conditions to delta-G-degree-prime?,"delta-G = delta-G-degree-prime + RT ln(Q), where Q is the mass-action ratio."

Bioenergetics,"What is the mass-action ratio Q, and how does it differ from Keq?","Q = [products]/[reactants] at any given (not necessarily equilibrium) moment, using actual concentrations; Keq is specifically the ratio at equilibrium."

Bioenergetics,How do cells drive a thermodynamically unfavorable reaction (delta-G > 0) forward?,"By coupling it to a strongly favorable (exergonic) reaction, such as ATP hydrolysis, so that the summed delta-G of the coupled reactions is negative."

Bioenergetics,"Why might a thermodynamically favorable reaction (delta-G < 0) not actually proceed at a noticeable rate (e.g., diamond to graphite)?","Because thermodynamics only describes favorability/direction, not rate; the reaction may have a large activation energy barrier (delta-G-double-dagger), making it kinetically very slow."

Bioenergetics,What can lower a reaction's activation energy barrier without changing delta-G?,"A catalyst, such as an enzyme."

Bioenergetics,"The isomerization of Glucose-6-P to Fructose-6-P has delta-G-degree-prime = +1.7 kJ/mol, yet proceeds forward in glycolysis. How is this possible?","Because actual cellular concentrations of glucose-6-P and fructose-6-P are not 1 M each; using delta-G = delta-G-degree-prime + RT ln(Q) with the real (low, unequal) concentrations gives a negative delta-G, making the reaction favorable under cellular conditions."

Bioenergetics,Convert a C-C bond energy of 350 kJ/mol into kcal/mol.,About 83.7 kcal/mol (350 / 4.184 kJ per kcal).

Bioenergetics,What is the approximate length of a C-C bond?,About 1.5 Angstroms (0.154 nm).

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Alanine?",Three-letter: Ala; One-letter: A; Functional group: alkane.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Cysteine?",Three-letter: Cys; One-letter: C; Functional group: thiol.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Aspartate (aspartic acid)?",Three-letter: Asp; One-letter: D; Functional group: carboxylic acid.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Glutamate (glutamic acid)?",Three-letter: Glu; One-letter: E; Functional group: carboxylic acid.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Phenylalanine?",Three-letter: Phe; One-letter: F; Functional group: phenyl.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Glycine?",Three-letter: Gly; One-letter: G; Functional group: hydrogen.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Histidine?",Three-letter: His; One-letter: H; Functional group: imidazole.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Isoleucine?",Three-letter: Ile; One-letter: I; Functional group: alkane.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Lysine?",Three-letter: Lys; One-letter: K; Functional group: amine.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Leucine?",Three-letter: Leu; One-letter: L; Functional group: alkane.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Methionine?",Three-letter: Met; One-letter: M; Functional group: thioether.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Asparagine?",Three-letter: Asn; One-letter: N; Functional group: amide.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Proline?",Three-letter: Pro; One-letter: P; Functional group: (cyclic) alkane.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Glutamine?",Three-letter: Gln; One-letter: Q; Functional group: amide.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Arginine?",Three-letter: Arg; One-letter: R; Functional group: guanidinium.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Serine?",Three-letter: Ser; One-letter: S; Functional group: alcohol.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Threonine?",Three-letter: Thr; One-letter: T; Functional group: alcohol.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Valine?",Three-letter: Val; One-letter: V; Functional group: alkane.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Tryptophan?",Three-letter: Trp; One-letter: W; Functional group: indole.

Amino acids: identity & functional groups,"What is the three-letter code, one-letter code, and side chain functional group of Tyrosine?",Three-letter: Tyr; One-letter: Y; Functional group: phenol.

Amino acids: identity & functional groups,Which amino acid has the three-letter code Ala and one-letter code A?,Alanine (functional group: alkane).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Cys and one-letter code C?,Cysteine (functional group: thiol).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Asp and one-letter code D?,Aspartate (aspartic acid) (functional group: carboxylic acid).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Glu and one-letter code E?,Glutamate (glutamic acid) (functional group: carboxylic acid).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Phe and one-letter code F?,Phenylalanine (functional group: phenyl).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Gly and one-letter code G?,Glycine (functional group: hydrogen).

Amino acids: identity & functional groups,Which amino acid has the three-letter code His and one-letter code H?,Histidine (functional group: imidazole).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Ile and one-letter code I?,Isoleucine (functional group: alkane).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Lys and one-letter code K?,Lysine (functional group: amine).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Leu and one-letter code L?,Leucine (functional group: alkane).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Met and one-letter code M?,Methionine (functional group: thioether).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Asn and one-letter code N?,Asparagine (functional group: amide).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Pro and one-letter code P?,Proline (functional group: (cyclic) alkane).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Gln and one-letter code Q?,Glutamine (functional group: amide).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Arg and one-letter code R?,Arginine (functional group: guanidinium).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Ser and one-letter code S?,Serine (functional group: alcohol).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Thr and one-letter code T?,Threonine (functional group: alcohol).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Val and one-letter code V?,Valine (functional group: alkane).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Trp and one-letter code W?,Tryptophan (functional group: indole).

Amino acids: identity & functional groups,Which amino acid has the three-letter code Tyr and one-letter code Y?,Tyrosine (functional group: phenol).

Amino acids: identity & functional groups,Which amino acids have an alkane (simple hydrocarbon) side chain?,"Alanine, Isoleucine, Leucine, Valine (and Proline, whose side chain is a cyclic alkane)."

Amino acids: identity & functional groups,Which amino acids have an alcohol side chain?,Serine and Threonine.

Amino acids: identity & functional groups,Which amino acids have an amide side chain?,Asparagine and Glutamine.

Amino acids: identity & functional groups,Which amino acids have a carboxylic acid side chain?,Aspartate and Glutamate.

Amino acids: identity & functional groups,Which amino acid has a thiol side chain?,Cysteine.

Amino acids: identity & functional groups,Which amino acid has a thioether side chain?,Methionine.

Amino acids: identity & functional groups,"Which amino acid has a phenyl (benzene ring, no substituent) side chain?",Phenylalanine.

Amino acids: identity & functional groups,Which amino acid has a phenol side chain?,Tyrosine.

Amino acids: identity & functional groups,Which amino acid has an indole side chain?,Tryptophan.

Amino acids: identity & functional groups,Which amino acid has an imidazole side chain?,Histidine.

Amino acids: identity & functional groups,Which amino acid has a guanidinium side chain?,Arginine.

Amino acids: identity & functional groups,Which amino acid has an amine side chain?,Lysine.

Amino acids: identity & functional groups,Which amino acid's side chain is just a single hydrogen atom?,Glycine.

Amino acids: identity & functional groups,Which amino acid has a cyclic (secondary amine) structure where the side chain loops back to bond nitrogen?,Proline.

Amino acids: identity & functional groups,"Asparagine and Glutamine are the amide versions of which two acidic amino acids, respectively?",Asparagine = amide version of Aspartate (Asp/D); Glutamine = amide version of Glutamate (Glu/E).

Amino acids: identity & functional groups,What distinguishes Phenylalanine's side chain from Tyrosine's?,Tyrosine has an added hydroxyl group (phenol) on the aromatic ring; Phenylalanine's ring (phenyl) has no substituent.

Amino acids: identity & functional groups,What distinguishes Cysteine's side chain from Methionine's?,"Cysteine has a thiol (-SH) directly available for reactions/disulfide bonds; Methionine has a thioether (-S-CH3, sulfur bonded to two carbons), which is unreactive in that way."

Amino acids: pKa & charge,"List the 8 ionizable/titratable groups in proteins with their typical pKa (Table 3.1, textbook precise values).",Terminal alpha-carboxyl (C-term): 3.1; Asp/Glu: 4.1; His: 6.0; Terminal alpha-amino (N-term): 8.0; Cys: 8.3; Tyr: 10.9; Lys: 10.8; Arg: 12.5.

Amino acids: pKa & charge,List the 8 ionizable/titratable groups in proteins with their rounded/approximate pKa (course study guide values).,C-terminus: 3; Asp/Glu: 4; His: 6; N-terminus: 8; Cys: 8; Lys: 10; Tyr: 10; Arg: 12.

Amino acids: pKa & charge,What is the pKa of the terminal alpha-carboxyl group (C-terminus)?,~3 (precise value 3.1).

Amino acids: pKa & charge,What is the pKa of Asp and Glu side chains?,~4 (precise value 4.1).

Amino acids: pKa & charge,What is the pKa of the His side chain (imidazole)?,~6 (precise value 6.0).

Amino acids: pKa & charge,What is the pKa of the terminal alpha-amino group (N-terminus)?,~8 (precise value 8.0).

Amino acids: pKa & charge,What is the pKa of the Cys side chain (thiol)?,~8 (precise value 8.3).

Amino acids: pKa & charge,What is the pKa of the Tyr side chain (phenol)?,~10 (precise value 10.9).

Amino acids: pKa & charge,What is the pKa of the Lys side chain (amine)?,~10 (precise value 10.8).

Amino acids: pKa & charge,What is the pKa of the Arg side chain (guanidinium)?,~12 (precise value 12.5).

Amino acids: pKa & charge,"For an acidic-type group (carboxyl, thiol, phenol), which protonation state is charged?","The deprotonated form (e.g., -COO-, -S-, -O-) is charged (negative); the protonated form is neutral."

Amino acids: pKa & charge,"For a basic-type group (amine-based: Lys, Arg, His, N-terminus), which protonation state is charged?","The protonated form (e.g., -NH3+) is charged (positive); the deprotonated form is neutral."

Amino acids: pKa & charge,"General rule: if pH is below a group's pKa, which form dominates?",The protonated form dominates.

Amino acids: pKa & charge,"General rule: if pH is above a group's pKa, which form dominates?",The deprotonated form dominates.

Amino acids: pKa & charge,"At neutral pH, what is the charge state of Asp and Glu side chains, and why?","-1 (net negative) -- pH 7 is above their pKa (~4), so they are deprotonated (acidic-type groups are charged when deprotonated)."

Amino acids: pKa & charge,"At neutral pH, what is the charge state of Lys and Arg side chains, and why?","+1 (net positive) -- pH 7 is below their pKa (~10 and ~12), so they remain protonated (basic-type groups are charged when protonated)."

Amino acids: pKa & charge,"At neutral pH, what is the charge state of the His side chain, and why is it described as '~0 average' rather than a clean integer?","~0 average charge -- His's pKa (~6) is close to pH 7, so a meaningful fraction remains protonated (positive) even though the majority is deprotonated (neutral); this makes His useful as a proton donor/acceptor (e.g., in enzyme active sites)."

Amino acids: pKa & charge,"At neutral pH, what is the charge state of the Cys side chain, and why?","~0 average charge -- pH 7 is just below its pKa (~8), so it is mostly protonated (neutral thiol) with only a small deprotonated (thiolate, negative) fraction."

Amino acids: pKa & charge,"At neutral pH, what is the charge state of the Tyr side chain, and why?","0 (essentially neutral) -- pH 7 is well below its pKa (~10), so it remains almost entirely protonated (neutral phenol)."

Amino acids: pKa & charge,State the Henderson-Hasselbalch equation.,pH = pKa + log([A-]/[HA]).

Amino acids: pKa & charge,"According to Henderson-Hasselbalch, what is true when pH = pKa?",[A-] = [HA] -- the deprotonated and protonated forms are present in equal concentration.

Amino acids: pKa & charge,"Using Henderson-Hasselbalch, what is the ratio of deprotonated (thiolate) to protonated (thiol) cysteine at pH 6, given pKa(Cys) ~ 8?","log([A-]/[HA]) = pH - pKa = 6 - 8 = -2, so [A-]/[HA] = 10^-2 = 1:100 (protonated thiol form strongly dominates)."

Amino acids: pKa & charge,"A weak acid HA has Ka = 1.00 x 10^-5. If 0.100 mol is dissolved in 1 L water, what percent is dissociated at equilibrium?","About 1.00% (using Ka = x^2/C, x = sqrt(Ka x C) = sqrt(1e-5 x 0.1) = 1e-3 M, which is 1% of 0.1 M)."

Amino acids: pKa & charge,An amino acid derivative called aminoethanethiol is cysteine without the carboxylic acid (H2N-CH2-CH2-SH). What is its charge state at pH 4?,"+1 -- at pH 4 the amine (pKa ~8-10) is protonated (positive) and the thiol (pKa ~8) is also protonated (neutral), so net charge is +1."

Amino acids: pKa & charge,What is aminoethanethiol's charge state at pH 12?,"-1 -- at pH 12 the amine (pKa ~8-10) is deprotonated (neutral) and the thiol (pKa ~8) is deprotonated (negative, thiolate), giving a net charge of -1."

Isoelectric point (pI),What is the isoelectric point (pI) of a peptide?,The pH at which the peptide's net charge is exactly zero.

Isoelectric point (pI),What are the general steps for estimating a peptide's pI?,"1) List every titratable group (both termini + any D/E/H/C/Y/K/R side chains) with its pKa. 2) Sort pKa's ascending. 3) Starting at low pH, track net charge as you cross each pKa moving upward. 4) Find the two pKa's that bracket where net charge crosses zero. 5) pI = average of those two bracketing pKa's (or the pH where a table of computed net charges crosses zero)."

Isoelectric point (pI),"When a group's pKa is within 0.5 pH units of the pH being evaluated, what convention is used to assign its charge?","Assign a fractional charge of +0.5 or -0.5 (half of that group's fully charged value) instead of a full integer, since the group is roughly half protonated/half deprotonated."

Isoelectric point (pI),"For the peptide Ala-Glu-Arg-Phe-Asp-His (recurring recitation/exam peptide), what are its titratable groups and pKa's?",N-terminus: 8.0; Glu: 4.1; Arg: 12.5; Asp: 4.1; His: 6.0; C-terminus: 3.1.

Isoelectric point (pI),"For Ala-Glu-Arg-Phe-Asp-His, what is the net charge at pH 3?","+2.5 -- N-term +1, Glu 0 (protonated, pH well below pKa 4.1), Arg +1, Asp 0, His +1 (protonated, pH well below pKa 6.0), C-term -0.5 (pH 3 is within 0.5 units of its pKa 3.1, so fractional)."

Isoelectric point (pI),"For Ala-Glu-Arg-Phe-Asp-His, what is the net charge at pH 8?","-1.5 -- N-term +0.5 (pH exactly at its pKa 8.0, fractional), Glu -1, Arg +1, Asp -1, His 0 (deprotonated, pH well above pKa 6.0), C-term -1."

Isoelectric point (pI),"For Ala-Glu-Arg-Phe-Asp-His, what is the net charge at pH 11?","-2 -- N-term 0 (deprotonated), Glu -1, Arg +1 (still protonated, pH is 1.5 units below pKa 12.5), Asp -1, His 0, C-term -1."

Isoelectric point (pI),"For Ala-Glu-Arg-Phe-Asp-His, what is the estimated pI, and how do you find it?","Approximately pH 5. Stepping through pH values, net charge is +1 at pH 4 and exactly 0 at pH 5 (N-term +1, Glu -1, Arg +1, Asp -1, His +1, C-term -1 = 0), so the net charge crosses zero right at pH 5."

Isoelectric point (pI),"Two peptides, IHATEYANKEES and RARELIPSTICKK, are compared at pH 8. What is the net charge of each (using +/-0.5 for pKa's within 0.5 units)?",Peptide 1 (IHATEYANKEES): -2.5. Peptide 2 (RARELIPSTICKK): +2.

Isoelectric point (pI),Would peptides IHATEYANKEES and RARELIPSTICKK be expected to form an ionic interaction at pH 8? Why?,"Yes -- peptide 1 is negatively charged (-2.5) and peptide 2 is positively charged (+2) at pH 8, so they would be expected to attract electrostatically."

Isoelectric point (pI),"Which residues let peptide 2 (RARELIPSTICKK) interact favorably with DNA at pH 8, and why?",Its Arg and Lys residues -- they are positively charged at pH 8 and interact electrostatically with the negatively charged DNA phosphate backbone.

Isoelectric point (pI),How would you disrupt salt-bridge interactions between a DNA-binding protein and DNA to purify the protein (assuming pH ~7 must be maintained)?,Add salt (increase ionic strength) -- this is the same principle used as the elution condition in ion-exchange chromatography; high salt screens/out-competes the electrostatic interactions.

Isoelectric point (pI),"Roughly, at pH 2, what is the charge of free glycine (pKa's ~2.3 alpha-COOH, ~9.6 alpha-NH3+)?","+1 (both groups protonated: -COOH neutral, -NH3+ positive)."

Isoelectric point (pI),"Roughly, at pH 7, what is the charge of free glycine?",0 (zwitterion: -COO- negative and -NH3+ positive cancel out).

Isoelectric point (pI),"Roughly, at pH 12.5, what is the charge of free glycine?","-1 (both groups deprotonated: -COO- negative, -NH2 neutral)."

Isoelectric point (pI),"For free aspartic acid, what is the charge at pH 2, pH 7, and pH 12.5?","pH 2: +1; pH 7: -1; pH 12.5: -2 (both the alpha-carboxyl and the side-chain carboxyl are deprotonated, plus the alpha-amino group is deprotonated/neutral)."

Isoelectric point (pI),"For free arginine, what is the charge at pH 2, pH 7, and pH 12.5?","pH 2: +2; pH 7: +1; pH 12.5: -0.5 (near the guanidinium pKa of 12.5, so fractionally charged)."

Peptide bonds & primary structure,What is 'primary structure' of a protein?,The linear amino acid sequence.

Peptide bonds & primary structure,Is peptide bond hydrolysis or peptide bond formation more thermodynamically favorable?,"Hydrolysis is thermodynamically more favorable, but the reaction is extremely slow (the half-life of an amide bond is about 7 years) -- kinetics, not thermodynamics, keeps proteins stable."

Peptide bonds & primary structure,Why is the peptide (amide) bond considered 'rigid'/planar?,"Resonance delocalization of the nitrogen lone pair into the C=O pi system gives the C-N bond partial double-bond character, restricting rotation (barrier ~23 kcal/mol)."

Peptide bonds & primary structure,"Which is more stable, the cis or trans peptide bond configuration, and by how much?",Trans is more stable by about 8 kJ/mol (~2 kcal/mol) due to steric clash in the cis form.

Peptide bonds & primary structure,Why do X-Pro peptide bonds show a much higher proportion of cis-amide character (~10%) than typical X-X bonds?,"Proline's cyclic (secondary amine, pyrrolidine ring) structure means that even in the normally-favored trans configuration, there is steric clash between Pro's ring and the preceding residue; this greatly reduces the usual trans preference, so cis becomes far more common than for peptide bonds involving other residues."

Peptide bonds & primary structure,What is the 'omega' (w) dihedral angle?,"The dihedral angle around the peptide (amide) C-N bond itself, in addition to phi and psi; it is almost always ~180 degrees (trans) or, less commonly, ~0 degrees (cis)."

Peptide bonds & primary structure,"Which amino acid is most likely to show a different (cis) omega dihedral angle, and why is rotation around omega slower than around phi/psi?","Proline; omega rotation is restricted because the peptide bond has partial double-bond character (resonance), giving it a much higher rotational energy barrier than the single bonds involved in phi and psi."

Peptide bonds & primary structure,What is the phi (f) dihedral angle?,"The dihedral angle around the N-Calpha bond (backbone atoms C(carbonyl,i-1)-N-Calpha-C(carbonyl,i))."

Peptide bonds & primary structure,What is the psi (y) dihedral angle?,The dihedral angle around the Calpha-C(=O) bond (backbone atoms N-Calpha-C(carbonyl)-N(i+1)).

Peptide bonds & primary structure,What four consecutive atoms define the omega dihedral angle?,"Calpha-C(carbonyl)-N-Calpha, across the peptide bond itself."

Peptide bonds & primary structure,"Roughly what pH range of pKa's can be measured/exist appreciably in water, and why (the 'leveling effect')?","Roughly -1.74 to 14; an acid stronger than H3O+ or a base stronger than OH- cannot exist in appreciable concentration in water, because water itself will fully protonate/deprotonate them."

Peptide bonds & primary structure,Why does rotation around omega affect the rate of protein folding?,"Because cis/trans isomerization at omega (especially at X-Pro bonds) is slow, a protein synthesized with the 'wrong' omega isomer at a given position may fold slowly, needing to wait for isomerization (sometimes catalyzed by peptidyl-prolyl isomerase) before reaching the native state."

Ramachandran plot & dihedral basics,What does a Ramachandran plot show?,"The accessible (low-energy, sterically allowed) combinations of phi and psi backbone dihedral angles."

Ramachandran plot & dihedral basics,"On a Ramachandran plot, which two regions correspond to alpha-helix and beta-strand?","Alpha-helix: roughly phi = -60, psi = -45 (lower-left quadrant). Beta-strand: roughly phi = -120, psi = +120 (upper-left quadrant)."

Ramachandran plot & dihedral basics,"Which amino acid shows a significant distribution of positively-charged (unusual) phi angles on a Ramachandran plot, and why?","Glycine -- unlike all other amino acids, it has only a hydrogen atom as its 'side chain,' so it has no steric interference and can sample a much wider range of phi/psi space, including regions normally disallowed for other residues."

Ramachandran plot & dihedral basics,Why do Gly and Pro commonly appear in turns connecting beta-strands?,"Gly's lack of a side chain allows it the backbone flexibility (unusual phi/psi angles) required by tight turns; Pro's rigid ring pre-organizes the backbone into a fixed conformation and lacks an amide H, which is compatible with certain turn geometries."

Ramachandran plot & dihedral basics,What are the two general guidelines used to determine which secondary structures a peptide can form?,"(1) Avoid steric clashes (stay within Ramachandran-allowed phi/psi regions), and (2) maximize the number of hydrogen bonds."

Alpha helix,What backbone hydrogen bonding pattern defines the alpha-helix?,"Residue i's C=O hydrogen bonds to residue i+4's N-H (i, i+4 spacing), forming a 13-atom ring -- technically should be called a '4.13 helix.'"

Alpha helix,How many residues per turn does an alpha-helix have?,3.6 residues per turn.

Alpha helix,What handedness are almost all alpha-helices in natural proteins?,Right-handed (energetically favored due to less steric clash between side chains and backbone).

Alpha helix,"Which residues near the termini of an alpha-helix cannot form the standard i, i+4 hydrogen bond?",The first 3 N-H groups (at the N-terminus) and the last 3 C=O groups (at the C-terminus) of the helix.

Alpha helix,"Who predicted the structure of the alpha-helix, and when?","Linus Pauling, Robert Corey, and Herman Branson, in 1951 (before it was experimentally confirmed)."

Alpha helix,What residue spacing allows side chains to interact with each other on the same face of an alpha-helix?,"i, i+3 and i, i+4 spacing (side chains at these positions can form salt bridges, H-bonds, pi-stacking, or van der Waals contacts)."

Alpha helix,What is an 'amphipathic' alpha-helix?,A helix where one face (side) is predominantly hydrophobic and the opposite face is predominantly hydrophilic/polar.

Alpha helix,How is a helical wheel diagram constructed?,"Looking down the helix's central axis, each residue is placed sequentially around the wheel, advancing about 100 degrees per residue (consistent with 3.6 residues/turn); decreasing circle diameters can indicate residues farther along the helix."

Alpha helix,"Which amino acid has the highest alpha-helix propensity, and which two are the strongest 'helix breakers'?",Alanine has high helix propensity; Glycine and Proline are the classic helix breakers.

Alpha helix,Why is glycine a helix breaker?,"It is highly flexible (no side chain), so incorporating it into a helix costs a large conformational entropy penalty to restrict it to the narrow helical region of phi/psi space."

Alpha helix,Why is proline a helix breaker?,"It has no amide N-H to donate as a hydrogen bond (secondary amine), causing a gap in the helix's H-bond network, and its ring geometry creates steric clashes/distortions in the backbone."

Alpha helix,"How does Leucine's helix-stabilizing ability compare to Ile or Val, and why?","Leucine stabilizes helices more than Ile or Val; branching close to the backbone (as in Ile/Val, beta-branched) is less favorable (more crowding) than Leu's branching farther from the backbone."

Alpha helix,"What kinds of side-chain interactions at i,i+3 / i,i+4 spacing stabilize an alpha-helix?","Acid-base (salt bridge) interactions, e.g., Glu/Asp with Lys/Arg, and hydrophobic interactions between nonpolar residues."

Alpha helix,"In the peptide AARAAARAA (Arg at i and i+4), would you expect higher or lower helicity than (Ala)9, and why?","Lower helicity -- the two Arg side chains, positioned on the same face one helical turn apart, create a destabilizing electrostatic repulsion (both positively charged)."

Alpha helix,"In the peptide AAEAAARAA (Glu at i, Arg at i+4), would you expect higher or lower helicity than (Ala)9, and why?","Higher helicity -- the Glu and Arg side chains, positioned on the same face one helical turn apart, form a stabilizing electrostatic (salt bridge) interaction (opposite charges)."

Alpha helix,"In the peptide AARAAAWAA (Arg and Trp at i, i+4), would you expect higher or lower helicity than (Ala)9, and why?","Higher helicity -- Arg and Trp, positioned on the same face one helical turn apart, can form a stabilizing cation-pi interaction."

Alpha helix,"In the peptide AAVAAAVAA (Val substitutions), would you expect higher or lower helicity than (Ala)9, and why?",Lower helicity -- Valine is beta-branched and has a low intrinsic propensity to form an alpha-helix.

Alpha helix,"In the peptide AAGAAAGAA (Gly substitutions), would you expect higher or lower helicity than (Ala)9, and why?","Lower helicity -- Glycine has no side chain, is highly flexible, and is a classic 'helix-breaker.'"

Alpha helix,"Lysine has a relatively high alpha-helix propensity, but ornithine (one methylene group shorter) has much lower helix propensity. Why?","In ornithine, the side-chain amine (NH2) sits closer to the backbone than in lysine; this shorter side chain is more likely to disrupt/interfere with the helix, whereas amino acids that stabilize helices generally have their functional groups positioned farther from the backbone."

Alpha helix,A peptide of only Lysine residues has no defined structure at neutral pH but forms a stable alpha-helix near pH 11. Why?,"At neutral pH, all Lys side chains are protonated (+1), and the resulting strong electrostatic repulsion between adjacent positive charges prevents helix formation. Near pH 11 (above Lys's pKa ~10), the side chains become deprotonated/neutral, removing the repulsion and allowing the chain to fold into a stable helix."

Alpha helix,"What is the 3-10 helix, and what H-bond pattern defines it?","A tighter helix than the alpha-helix, with i, i+3 hydrogen bonding (10-atom ring); it is the second most common helix type after the alpha-helix."

Alpha helix,"What is the pi-helix, and what H-bond pattern defines it?","A wider helix than the alpha-helix, with i, i+5 hydrogen bonding (16-atom ring); rare in proteins."

Beta sheet,What are the consecutive dihedral angles characteristic of a beta-strand?,"Phi ~ -120 degrees, psi ~ +120 degrees."

Beta sheet,What is the approximate distance between adjacent amino acids along an extended beta-strand?,About 3.4 Angstroms (versus ~1.5 A rise per residue in an alpha-helix).

Beta sheet,Which amino acids are enriched in beta-strands?,"Branched amino acids (Val, Ile, Thr), aromatic amino acids (Tyr, Trp, Phe), and Cysteine."

Beta sheet,How do hydrogen bonds differ in orientation between parallel and antiparallel beta-sheets?,"In antiparallel sheets, H-bonds are directly across from each other (perpendicular to the strands, evenly spaced); in parallel sheets, H-bonds are angled/skewed, alternating between closer and farther spacing."

Beta sheet,"Which is generally more stable, parallel or antiparallel beta-sheets, and why?","Antiparallel beta-sheets are more stable, due to more linear (favorable) hydrogen bond geometry between strands."

Beta sheet,What does it mean that beta-sheets are 'pleated'?,"The sheet has a zig-zag, corrugated 3D shape (not flat) because alternating Calpha side chains point above and below the plane of the sheet."

Beta sheet,"What amphipathic pattern is often seen in beta-strands, and how does it differ from the pattern seen in alpha-helices?","An alternating H-P-H-P (hydrophobic-polar) pattern, since consecutive residues point to opposite faces of the sheet; this differs from the i,i+3/i,i+4 spacing pattern that creates amphipathicity in alpha-helices."

Beta sheet,A protein region shows the amino acid pattern -hydrophobic-polar-hydrophobic-polar-hydrophobic-polar-. What secondary structure does this most likely indicate?,"A beta-sheet (beta-strand), because the alternating H-P-H-P pattern matches residues alternating which face of the sheet they point toward."

Beta sheet,"What type of turn connects two adjacent antiparallel beta-strands, and which two amino acids are commonly found there?",A beta-turn (Type I or Type II); Glycine and Proline are common in turns because Gly provides backbone flexibility and Pro pre-organizes/constrains the turn.

Beta sheet,What distinguishes a Type I from a Type II beta-turn?,The orientation of the peptide plane between the second and third amino acid residues of the turn.

Beta sheet,"What is the Polyproline II (PPII) helix, and how many residues per turn does it have?",A left-handed helix formed by repeating proline residues in the trans-amide configuration; it has 3 residues per turn and is close in phi/psi space to the beta-sheet region of the Ramachandran plot.

Beta sheet,What distinguishes PPI from PPII helices?,"PPI arises from repeating cis-amide bonds (rare, generally only seen in organic solvents); PPII arises from repeating trans-amide bonds (much more common)."

Beta sheet,What characterizes a 'random coil' or disordered region of a protein?,No well-defined secondary or tertiary structure; the polypeptide rapidly interconverts between multiple conformations because energy differences between backbone conformations are small relative to thermal energy (kT).

Coiled coils,What is a coiled-coil structure?,"Two (or more) alpha-helices that wrap around each other in a left-handed superhelical twist, stabilized by hydrophobic packing."

Coiled coils,"What is the 'heptad repeat' in a coiled-coil, and which positions are hydrophobic?",A 7-residue repeating pattern (labeled a-b-c-d-e-f-g); the a and d positions are hydrophobic and pack together at the helix-helix interface.

Coiled coils,"How many residues per turn does a coiled-coil helix have, and how does this differ from a standard alpha-helix?",About 3.5 residues per turn (versus 3.6 for a standard alpha-helix); this slight difference requires the helices to tilt/supercoil around each other to maintain optimal packing.

Coiled coils,"Besides hydrophobic packing at a/d, what other interaction can stabilize a coiled-coil?",An ion-ion salt bridge between residues at the e and g positions of the heptad repeat.

Coiled coils,"A 50-residue protein appears larger than expected by size-exclusion chromatography, and is found to be mostly helical, with the sequence ---LQTMDSRLSTLGQN---. What structure explains this?","A coiled-coil dimer (or amphipathic helix dimer) -- the hydrophobic residues (3 Leu and 1 Met) fall at the 'a' and 'd' heptad positions, driving dimerization through hydrophobic packing, which makes the protein behave as if it were larger."

Coiled coils,"In that same sequence, would replacing the Arg residue with Glu weaken or strengthen the coiled-coil dimer?","Weaken it -- Arg forms a stabilizing salt bridge with a Glu at the complementary e/g heptad position; replacing Arg with Glu would create charge repulsion (Glu-Glu) instead, destabilizing the structure."

Coiled coils,What is the difference between a parallel and an antiparallel coiled-coil in terms of heptad register?,Parallel: a/a and d/d positions pack together (same positions on each helix). Antiparallel: a'/d and a/d' positions pack together (offset registers between the two helices).

Tertiary & super-secondary structure,What is a 'protein domain'?,A part of a polypeptide chain that is independently stable (folds on its own) and often has a distinct function.

Tertiary & super-secondary structure,Name several common 'super-secondary structure' motifs.,"Helix-turn-helix, helix-loop-helix, beta-alpha-beta, Greek key, and beta-barrel."

Tertiary & super-secondary structure,What are the four major classes of tertiary structure (by secondary structure content)?,"All-alpha (e.g., myoglobin), all-beta (e.g., OmpF porin), alpha/beta (e.g., lipase), and mixed alpha+beta (e.g., tyrosine kinase)."

Tertiary & super-secondary structure,"For a typical water-soluble globular protein, where are hydrophobic and hydrophilic residues generally located?",Hydrophobic residues are typically buried in the interior; hydrophilic/charged residues are typically on the solvent-exposed surface.

Tertiary & super-secondary structure,"Why doesn't the 'hydrophobic-in, hydrophilic-out' rule apply straightforwardly to a membrane protein like rhodopsin?","Because much of a membrane protein's surface is in contact with the hydrophobic lipid bilayer rather than water, so hydrophobic residues face outward (toward lipid) in the membrane-spanning regions, while polar/charged residues may be found in the interior or in loops exposed to the aqueous environment."

Tertiary & super-secondary structure,What common finding governs how alpha-helices and beta-sheets pack together in tertiary structure?,"Helices and sheets tend to pack in separate layers, because their backbone H-bonding geometries cannot align between the two types of structure; hydrophobic interactions between these layers are a key stabilizing force."

Tertiary & super-secondary structure,What is the Rossmann fold?,"A common protein fold that combines alternating alpha-helices and beta-strands (e.g., seen in lactate dehydrogenase), often used for nucleotide binding."

Tertiary & super-secondary structure,Are polypeptide segments that are close together in primary sequence usually close together in the folded tertiary structure?,Yes -- this is a common finding in tertiary structure.

Tertiary & super-secondary structure,In what handed sense are beta-sheets most stable when twisted?,Right-handed twist.

Fibrous proteins,"What general structural role do fibrous proteins play, and what shape do they have?","They have a filamentous/elongated form and play structural roles in animal cells and tissues (e.g., skin, connective tissue, hair, silk)."

Fibrous proteins,"What amino acids dominate collagen, and what is its repeating sequence motif?","Glycine, Proline, and Hydroxyproline (Hyp); the repeating motif is Gly-X-Y, where X is often Pro and Y is often Pro or Hyp."

Fibrous proteins,Why must glycine occur at every third position in collagen's triple helix?,Because the three polypeptide chains pack extremely tightly together in the triple helix; only glycine's single H side chain is small enough to fit at the tightly-packed core (interior) position of the helix without causing a steric clash.

Fibrous proteins,What is the basic structural unit of a collagen fiber?,"Tropocollagen -- a triple helix of three polypeptide chains, about 1000 residues long."

Fibrous proteins,Describe the helical handedness of collagen at the individual-chain and triple-helix levels.,Each individual chain forms a left-handed helix (~3.3 residues/turn); the three chains then wrap around each other in a right-handed superhelical twist.

Fibrous proteins,What stabilizes the collagen triple helix?,Hydrogen bonds between the three polypeptide chains (in addition to the tight steric packing enabled by Gly at every third position).

Fibrous proteins,What amino acid composition and secondary structure characterize silk fibroin?,"Almost half glycine, with alanine or serine making up most of the rest, in a repeating sequence like [Gly-Ala-Gly-Ala-Gly-Ser-...]; it forms long regions of antiparallel beta-sheet with chains running parallel to the fiber axis."

Fibrous proteins,What secondary structure and overall architecture characterizes alpha-keratin?,"Predominantly alpha-helical, built on a coiled-coil (dimer) structure; keratins are part of the broader intermediate filament protein family."

Fibrous proteins,"Match the amino acid group to the fibrous protein: Glycine, Proline, Hydroxyproline.",Collagen.

Fibrous proteins,"Match the amino acid group to the fibrous protein: Glycine, Alanine, Serine.",Silk (silk fibroin).

Fibrous proteins,"What gives collagen its high glycine content functional importance, compared to silk's high glycine content?","In collagen, Gly is needed for tight triple-helix packing (steric requirement at every 3rd position); in silk, Gly (alternating with Ala/Ser) allows tight interdigitation/stacking of antiparallel beta-sheets."

Quaternary structure,What is 'quaternary structure'?,The association of multiple folded polypeptide chains (subunits) into a specific multi-subunit complex.

Quaternary structure,What types of non-covalent (and covalent) interactions stabilize quaternary structure?,"The same interactions that stabilize tertiary structure: salt bridges, hydrogen bonding, van der Waals forces, the hydrophobic effect, plus (sometimes) covalent disulfide bonds."

Quaternary structure,Give three reasons why proteins form quaternary structures.,"(1) New structural properties (especially in fibrous proteins), (2) improved regulation (allostery), (3) linking functions in close proximity (e.g., multi-enzyme complexes)."

Quaternary structure,What is the difference between a hetero-oligomer and a homo-oligomer?,"A hetero-oligomer is composed of different (non-identical) subunit types (e.g., hemoglobin, a heterotetramer); a homo-oligomer is composed of identical subunits (e.g., a homodecamer)."

Protein folding,What did Anfinsen's ribonuclease refolding experiment demonstrate?,"That the information determining a protein's secondary and tertiary structure is contained entirely in its amino acid sequence -- proteins can spontaneously refold to their native, active conformation after being fully unfolded/denatured (using urea and beta-mercaptoethanol), without any additional cellular machinery."

Protein folding,"In Anfinsen's experiment, what did urea do, and what did beta-mercaptoethanol (BME) do?","Urea denatured/unfolded the protein (disrupted non-covalent interactions); BME reduced the disulfide bonds (broke the covalent S-S crosslinks), fully unfolding ribonuclease into a random coil."

Protein folding,What does Anfinsen's work imply about the relationship between a protein's native structure and its free energy?,The native (folded) three-dimensional structure corresponds to the protein's global free energy minimum under physiological conditions.

Protein folding,"Since you 'cannot unfry an egg white' (egg white proteins can't easily be refolded after cooking), does this disprove Anfinsen's hypothesis? Why or why not?","No -- proteins in a fried egg are denatured into the unfolded state, but they either (1) become too concentrated (aggregate) or (2) would require molecular chaperones to refold to their native state; the thermodynamic native state is still the free energy minimum, but the unfolded/aggregated proteins are kinetically trapped and cannot easily reach it under these conditions."

Protein folding,What is Levinthal's paradox?,"The observation that a protein has an astronomically large number of possible conformations (e.g., ~3^100 for a 100-residue protein), so a random exhaustive search would take far longer than the age of the universe -- yet real proteins fold in microseconds to milliseconds. This means folding cannot occur via random search."

Protein folding,How is Levinthal's paradox resolved?,"Folding is not a random search; it follows a funneled energy landscape where partially-correct folding intermediates are progressively stabilized, guiding the protein efficiently toward the native state (often via fast local folding of secondary structure elements first, then assembly into the global fold)."

Protein folding,What does a 'folding funnel' energy landscape diagram represent?,How conformational entropy decreases and stability increases as the protein moves from many unfolded conformations at the wide top of the funnel toward the single native conformation at the narrow bottom (lowest free energy).

Protein folding,What does a 'rugged' (vs. smooth) folding energy landscape imply about folding kinetics?,"A rugged landscape has multiple local energy minima/barriers, meaning the protein can fold through multiple pathways and may become transiently trapped in partially-folded intermediates, affecting folding rate/complexity."

Protein folding,"What are the two most important stabilizing forces in protein folding, and what is the single most important destabilizing force?","Stabilizing: (1) the hydrophobic effect, (2) hydrogen bonding. Destabilizing: loss of conformational entropy of the protein chain upon folding."

Protein folding,List five forces/factors that drive protein folding (per Dill & MacCallum).,"(i) Hydrogen bonds, (ii) van der Waals interactions, (iii) backbone dihedral angle preferences, (iv) electrostatic interactions, (v) hydrophobic interactions."

Protein folding,What does 'protein folding is cooperative' mean?,"Folding/unfolding shows a steep, 'all-or-none' transition (e.g., with temperature or denaturant) rather than a gradual one -- consistent with folding being a two-state process (Unfolded <-> Native) for many proteins."

Protein folding,"What confirmed that a chemically synthesized protein (made without any cellular machinery) still folds correctly, further supporting Anfinsen's hypothesis?","Bruce Merrifield's chemical synthesis of ribonuclease A, which showed that a synthetic polypeptide (built purely by chemistry, no ribosomes/chaperones) could fold into a catalytically active enzyme."

Protein folding,What surprising experiment involved a protease made entirely from D-amino acids?,"A 'D-enzyme' (protease synthesized entirely from D-amino acids) was made and shown to fold and function (mirror-image catalysis on mirror-image, D-amino-acid substrates), further supporting that sequence (and its chirality) alone determines structure/function."

Protein folding,"What are molecular chaperones, and when are they needed?","Proteins that assist other proteins in reaching their native folded state; needed by proteins that would otherwise become kinetically trapped in misfolded/aggregated states (i.e., the thermodynamic native state exists, but cannot be reached unaided)."

Protein folding,Does the need for chaperones disprove the thermodynamic hypothesis of protein folding (that the native state is the global free energy minimum)? Explain.,No (False) -- chaperones don't change the thermodynamic native state; they simply help kinetically-trapped/misfolded proteins escape local energy minima ('kinetic traps') so they can reach the true global free energy minimum (their native state).

Protein folding,Name three examples of protein chaperones/folding-helper systems.,"Chaperonins (e.g., GroEL/GroES), Hsp70 (e.g., DnaK), and Hsp90; also protein disulfide isomerase (for Cys/cystine) and peptidyl-prolyl isomerase (for amide cis/trans isomerization)."

Protein folding,How does the GroEL/GroES chaperonin system assist folding?,"It encloses a single non-native (partially folded) protein within a central cavity, isolating it from other proteins (preventing aggregation) while ATP-driven conformational changes create an environment conducive to proper folding; it folds roughly 10% of the E. coli proteome."

Protein folding,How does the Hsp70/DnaK chaperone system work?,"A partially unfolded substrate binds in a cleft between DnaK's beta-sandwich domain and an alpha-helical 'lid'; ATP hydrolysis to ADP causes the lid to close/swing down, providing a protected environment for the substrate to refold before release."

Protein folding,"An Ile residue buried in a protein's hydrophobic core is mutated to Lys. Would you expect this to cause a structural change, and why?","Yes -- Lys is positively charged and hydrophilic at pH 7, while Ile is uncharged/hydrophobic; placing a charged residue in the greasy, water-excluded core is highly destabilizing and would likely disrupt the fold."

Protein folding,Why is the chaperone trigger factor found in very high concentrations (up to 50 uM) in bacterial cells?,Because a very large number of nascent polypeptides are continuously being synthesized by ribosomes and require trigger factor's assistance as they emerge from the ribosome.

Protein folding diseases,What is the general difference between a 'gain of function' and 'loss of function' protein misfolding disease?,"Loss of function: the misfolded protein can no longer perform its normal job. Gain of function: the misfolded protein acquires a new, often toxic, activity (e.g., forming aggregates that damage cells) -- amyloid diseases are typically gain-of-function."

Protein folding diseases,What secondary structure dominates amyloid fibrils?,"Beta-sheet -- amyloid fibrils are beta-sheet-rich, cross-beta structures."

Protein folding diseases,What is the 'cross-beta' structure of an amyloid fibril?,"An arrangement where beta-strands run perpendicular to the long fibril axis (so the sheets' beta-strands stack along the fibril), and two sheets pack together to form the fibril's spine."

Protein folding diseases,What makes prion disease unusual compared to other protein misfolding diseases?,"It is transmissible/infectious -- the misfolded (scrapie) form of the prion protein itself can act as a template to convert normal prion protein into the misfolded form, without any nucleic acid involved."

Protein folding diseases,"What protein is implicated in Alzheimer's disease, and what does it do?",Beta-amyloid; it misfolds and forms aggregates (amyloid fibrils/plaques).

Protein purification,What does the acronym CHASM stand for in protein purification?,"Charge, Hydrophobicity (solubility), Affinity, Stability, Molecular weight (size) -- the physical properties used to design purification strategies."

Protein purification,Which type of chromatography separates proteins based on charge?,Ion-exchange chromatography.

Protein purification,Which type of chromatography separates proteins based on size?,Size-exclusion (gel-filtration) chromatography.

Protein purification,"Which type of chromatography separates proteins based on a specific binding interaction (e.g., antibody-antigen, enzyme-substrate mimic)?",Affinity chromatography.

Protein purification,How would you use a protein's differing thermal stability to separate it from a contaminating protein?,"Heat the mixture to a temperature where the less stable (lower-melting) contaminant denatures/precipitates/aggregates while the target protein (higher melting temperature) remains folded and soluble, then remove the precipitate (e.g., by centrifugation)."

Protein purification,"Histone proteins are rich in Arginine and Lysine. What type of ion-exchange resin would best purify them, and why?","A cation-exchange (negatively charged) resin -- Arg and Lys side chains are positively charged at neutral pH, so a negatively charged resin will bind them, while proteins lacking this excess positive charge will not bind as strongly."

Protein purification,"An enzyme (24 kDa, pI 5.5) is contaminated with a similarly-sized protein (pI 7.0) and a much larger protein (100 kDa, pI 5.4). Outline a CHASM-based purification strategy.","First, use ion-exchange chromatography to separate the target enzyme (pI 5.5) from the similarly-sized contaminant (pI 7.0), since they have different charge properties at a given pH. Then use size-exclusion (gel-filtration) chromatography to separate the target from the 100 kDa contaminant (different size) -- this step also removes salt from the ion-exchange elution."

Protein purification,"In an evaluation of a purification protocol, why does 'total units of activity' decrease at each step while 'specific activity' increases?","Some of the target protein is inevitably lost at each purification step (lowering total activity/yield), but the fraction of non-target ('contaminating') protein is being removed even faster, so the activity per milligram of total protein (specific activity) rises -- indicating increasing purity."

Protein purification,"What is SDS-PAGE used for, and what does SDS do?","It separates proteins primarily by size (molecular weight); SDS (a detergent) denatures proteins and coats them with a uniform negative charge, so migration through the gel depends mainly on size, not native charge/shape."

Protein purification,"In an SDS-PAGE experiment comparing a protein with/without beta-mercaptoethanol (BME) and/or a protease (P1), what does a shift in migration in the +BME lane (relative to no BME) indicate?","That the protein contains at least one disulfide bond; BME reduces/cleaves the disulfide, and if this releases the protein into unequal-sized pieces or changes its apparent size, it indicates where the disulfide links the chain(s)."

Protein purification,"If BME cleavage of a single-chain protein's internal disulfide releases two unequal-length fragments, what does this tell you about the disulfide's location?",The disulfide bond is not in the exact middle of the protein (since the two resulting pieces are of different sizes).

Protein structure determination,What are the three major experimental methods for determining protein structure discussed in this course?,"X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and Cryo-electron microscopy (cryo-EM)."

Protein structure determination,What is the primary requirement/limitation of X-ray crystallography?,"The protein must be crystallized (requires a large amount of pure, conformationally homogeneous protein); it gives a static picture and struggles with membrane proteins."

Protein structure determination,What are the advantages of X-ray crystallography?,High resolution and direct visualization of atom placement.

Protein structure determination,What is the primary size limitation of protein NMR?,Proteins larger than about 300 residues are very difficult to solve by NMR.

Protein structure determination,What are the advantages of protein NMR?,"Non-perturbing (isotope) labeling, potential atomic-level resolution (~1 A or better), samples can be studied under functional/solution conditions, and it can capture protein dynamics."

Protein structure determination,What is the primary size limitation of cryo-EM (opposite to NMR's limitation)?,"Cryo-EM has a lower size limit -- proteins/particles that are too small scatter electrons too weakly (low contrast), making them hard to image; bigger particles work better."

Protein structure determination,Why can't a very high electron flux/dose be used in cryo-EM?,It would cause radiation damage to the biological sample.

Protein structure determination,What are the advantages of cryo-EM?,"No need for crystallization (proteins imaged as single particles in near-native, vitrified conformations), effective for large complexes and membrane proteins, and electrons in principle allow very high resolution."

Protein structure determination,What are disadvantages of cryo-EM?,"Typically destroys the sample, small/flexible proteins remain difficult, requires high sample purity/homogeneity, expensive instrumentation, and often poor signal-to-noise (noisy images)."

Protein structure determination,"In a high-resolution (1.8 A) cryo-EM structure, a 5-residue loop connecting two alpha-helices shows very weak density. How can part of an otherwise well-resolved structure show poor density?","That region must not have existed in a single, unique conformation -- i.e., it is conformationally heterogeneous (dynamic or statically disordered across particles), which averages out/blurs its density in the reconstruction, even though the rest of the well-ordered structure resolves clearly."

Protein structure determination,"Compared to X-ray crystallography, what is the primary advantage of cryo-EM?",Proteins do not need to be crystallized.

Protein structure determination,"Compared to NMR, what is the primary disadvantage of cryo-EM (the 'opposite' limitation)?","A size/contrast problem -- cryo-EM struggles with small biomolecules (too little electron scattering/contrast, and radiation damage limits electron dose), whereas NMR struggles with large ones."