UWorld Biochemistry Amino Acids and Proteins: Quiz #1

• Bonds formed with energy input, like peptide bonds via GTP hydrolysis, are thermodynamically unstable but kinetically stable if they break slowly without an enzyme.

Ion-Exchange Chromatography

• Proteins bind to columns with opposite charges in ion-exchange chromatography.
• Anion-exchange chromatography uses positively charged beads to bind negatively charged proteins (pI below the working pH).
• Cation-exchange chromatography uses negatively charged beads to bind positively charged proteins (pI above the working pH).
• In cation exchange chromatography, the pH must be lower than the lowest pKa of all amino acids to ensure they are fully protonated and positively charged for binding.
• In ion-exchange chromatography, proteins bind to the column in low-salt conditions, as salt ions compete for binding sites.
• High salt is used to elute proteins by displacing them from the column.

Affinity Chromatography

• Affinity chromatography separates proteins by binding strength to a ligand (kD).
• Proteins with similar binding affinities will not separate effectively using affinity chromatography.
• In tagged protein experiments, only the tag that matches the capture antibody (e.g., Flag with α-Flag) binds directly to the column; any other retained protein must be interacting with the bound protein, not the column itself.

Protein Charge and Isoelectric Point (pI)

• A protein's charge depends on its isoelectric point (pI) at a given pH.
• If pH < pI, the protein is positively charged and binds to cation-exchange columns.
• If pH > pI, the protein is negatively charged and binds to anion-exchange columns.
• pH < pI means the protein is positively charged.
• pH > pI means the protein is negatively charged.
• When pH = pI, proteins are neutral, least soluble, and least stable, making them likely to aggregate and function poorly.
• A protein’s experimental pI can differ from its theoretical pI due to posttranslational modifications or electrostatic interactions in the folded structure, which alter pKa values; however, the subcellular pH environment or starting pH in IEF does not affect the final pI measurement.
• A protein is electrically neutral when the ambient pH equals its isoelectric point (pI).

Size-Exclusion Chromatography

• Size-exclusion chromatography separates molecules by size, with larger molecules eluting first.
• Proteins with similar molecular weights will not separate effectively using size-exclusion chromatography.
• In SEC, larger proteins migrate faster.

Alpha Helices

• Alpha helices are stabilized by hydrogen bonds within the peptide backbone.
• Secondary structure consists of α-helices and β-sheets, stabilized by hydrogen bonds between backbone atoms.

Tertiary Structures and Transmembrane Helices

• Hydrophobic interactions stabilize tertiary structures and transmembrane helices.
• Tertiary structures are primarily driven by the hydrophobic effect, where nonpolar residues are buried to avoid water.
• Proteins lacking hydrophobic amino acids have a weaker hydrophobic effect and are less likely to fold into a defined three-dimensional shape.
• Amino acids like serine and arginine have polar or charged side chains that allow them to form hydrogen bonds with water or other polar molecules, supporting solubility and interaction in aqueous environments.

Disulfide Bonds

• Disulfide bonds are covalent interactions between cysteine residues that stabilize tertiary structure.
• Disulfide bonds form through oxidation, increased by oxidizing agents such as oxygen, which promotes dimerization.
• Acids and bases do not promote disulfide bond formation.
• Reducing agents break disulfide bonds and decrease dimerization.
• Disulfide bonds form in oxidizing environments like the endoplasmic reticulum or extracellular space, but not in the cytosol, which is a reducing environment.
• Nonreducing SDS-PAGE does not break disulfide bonds, so protein subunits held together by disulfide linkages remain connected; reducing agents are required to separate such subunits.
• A reducing agent breaks disulfide bonds, revealing the true monomer size. Disulfide-linked dimers appear larger when a reducing agent is absent in both methods.

Protein Structure Stabilization

• Side chain interactions contribute to tertiary structure stabilization.
• Secondary structures (alpha helices, beta sheets) are stabilized by hydrogen bonding within the peptide backbone.
• Amino acids like serine and arginine have polar or charged side chains that allow them to form hydrogen bonds with water or other polar molecules, supporting solubility and interaction in aqueous environments.
• Hydrophilic residues, like those in reflectins, form hydrogen bonds with water, which helps them stay soluble but do not drive protein folding.

Transmembrane Helices Composition

• Transmembrane helices are composed primarily of hydrophobic amino acids.
• A region with a hydrophobicity value above 0 is considered hydrophobic and is more likely to interact with the hydrophobic core of membranes or be buried in a folded protein if folding occurs.

His-Tag Affinity Chromatography

• In His-tag affinity chromatography, the histidine side chain (imidazole ring) binds Ni²⁺ in the column.
• Imidazole is used to elute the protein, competing for Ni²⁺ binding without denaturation.

Phosphorylation Effects

• Phosphorylation lowers the isoelectric point (pI) by adding negatively charged phosphate groups.
• For phosphorylation to occur, ATP is needed to provide the phosphate group and energy for the reaction.
• Phosphorylation typically occurs on amino acids with hydroxyl groups—mainly serine, threonine, and tyrosine—because these groups act as nucleophiles in kinase-catalyzed reactions.

Enzyme Activity and Cooperativity

• A sigmoidal curve in enzyme activity indicates positive cooperativity.
• Regulatory proteins likely strengthen cooperative interactions rather than causing dissociation.
• Enzymes need their 3D shape (especially quaternary structure) to function; without that structure, there is no active site and no enzymatic activity.

Enzyme Regulation

• If a regulatory protein fully dissociates from an enzyme upon modification, the enzyme’s activity should return to baseline.
• A molecule is an allosteric activator if it binds to a site other than the active site and causes a conformational change that increases the protein’s function.

Effects of High Temperatures on Proteins

• High temperatures can disrupt subunit interactions (quaternary) and unfold proteins (tertiary).

Proline as a Unique Amino Acid

• Proline's R group forms a ring with the backbone amino group, making it a secondary amine.
• Peptide bonds in all amino acids are planar due to resonance.
• Proline is rigid and found in β-turns.

BN-PAGE

• BN-PAGE is ideal for studying multimeric proteins, maintaining their native structure.
• If SDS-PAGE reveals only one band, the complex consists of identical subunits (homo-multimer).
• Multiple bands on SDS-PAGE indicate different subunits (hetero-multimer).
• Coomassie dye ensures all proteins migrate toward the anode without denaturing them.
• Native PAGE preserves protein structure and allows multimers to migrate together, but knowing the migration pattern alone does not explain whether the protein retains its function.
• SDS denatures higher-order protein structure (2°, 3°, 4°) but does not disrupt primary structure, because it does not break peptide bonds between amino acids.

Determining Secondary Structure

• The predominant secondary structure is determined by comparing the entire spectrum.

Pore Radius Determination

• The pore radius is determined by when conductance stops decreasing and returns to baseline.

Specific Activity and Purity

• Highest Specific Activity = Highest Purity.
• High salt (NaCl) disrupts electrostatic interactions (like salt bridges) in proteins, leading to denaturation and loss of enzyme activity, which reduces NADH production.
• NaCl is a neutral salt that does not enhance hydrogen bonding, alter protein hydrophobicity, or change solution basicity; instead, it disrupts electrostatic interactions like salt bridges, which can denature proteins and reduce their function.

GTPase-Activating Proteins (GAPs)

• GTPase-activating proteins (GAPs) activate GTP hydrolysis.
• GAPs inhibit G protein signaling by converting GTP to GDP.

Ras and RASAL in Signaling

• Ras activates signaling when GTP-bound.
• RASAL inhibits signaling by accelerating GTP hydrolysis.

Glycine Chirality

• Glycine is achiral because its α-carbon is bonded to two hydrogens.
• Standards for biological samples should use L-forms of naturally occurring amino acids (except glycine, which is achiral).
• Enzymes like proteases are chiral and typically act only on peptides made from ʟ-amino acids, not ᴅ-amino acids.

G12C Protonation

• G12C is less protonated than free cysteine at pH 7.0.

Aromatic Rings

• Aromatic rings contain conjugated pi bonds.

pH Dependence

• Steps requiring a specific charged form are pH-dependent, while simple binding is usually pH-independent.
• When a question asks about protonation, use the pKa to determine the answer—groups are protonated when pH is below pKa and deprotonated when pH is above pKa.
• At pH 7, the carboxyl group should be COO⁻, not COOH.

Steric Hindrance

• Amino acids with large side chains cause more steric hindrance.
• Tryptophan has the largest and bulkiest side chain of all amino acids.

Mutation Effects on Isoelectric Point

• A mutation that removes a negatively charged residue increases the isoelectric point (pI).

Peptide Properties

• Peptides with the same amino acid composition have the same isoelectric point and hydrophobicity.
• Different sequences mean they have different primary structures.
• Forming a peptide bond is a condensation reaction where water is released.
• Breaking a peptide bond is a hydrolysis reaction where water is added.
• The N-terminus has a positive charge (–NH₃⁺).
• The C-terminus has a negative charge (–COO⁻).
• In peptide synthesis, the carboxyl group of the incoming amino acid acts as an electrophile and reacts with the amino group (nucleophile) of the growing chain.
• In solid-phase peptide synthesis, the peptide is built from the C-terminus to the N-terminus, with each new amino acid added by reacting its carboxyl group (electrophile) with the amino group (nucleophile) at the N-terminus of the growing chain.
• The C-terminus (carboxyl group) of the incoming amino acid acts as the electrophile, and the N-terminus (amino group) of the growing peptide chain is the nucleophile that attacks it to form a peptide bond.
• Primary structure is the amino acid sequence from N-terminus to C-terminus and can only be altered by breaking peptide bonds or by changing the identity of one or more amino acids within the original sequence (such as by mutation).
• When secondary and tertiary structures are absent, a protein’s structure is primarily defined by its primary structure, which consists of a specific sequence of amino acids linked by covalent peptide bonds.
• Primary structure is the sequence of amino acids linked by covalent peptide bonds.
• Primary structure is stabilized by peptide bonds, which are covalent bonds linking amino acids in a linear sequence.
• Schiff base formation requires a nucleophilic primary amine, which reacts with a carbonyl group (like an aldehyde) to form an imine linkage.

Protonation Ratios

• pH is 1 unit below pKa → 10:1 ratio of protonated:deprotonated.
• pH is 1 unit above pKa → 1:10 ratio of protonated:deprotonated.
• pH < pKa → more protonated.
• pH > pKa → more deprotonated.
• Ionizable amino acids show 3 buffer regions on a titration curve—one for the C-terminus, one for the N-terminus, and one for the side chain (if applicable).
• Lysine's side chain has a pKa of 10.5, giving it a third buffer region above pH 10.

Antibodies and Epitopes

• Antibodies bind protein epitopes using noncovalent protein-protein interactions.
• Linear epitopes come from the primary structure and are not affected by denaturation, and conformational (folded) epitopes depend on the protein’s 3D shape and are lost when the protein is denatured by SDS.

Comparing Peptide/Protein Properties

• When comparing peptides or proteins, check for net charge differences, especially with amino acid sequences.
• Peptides with the same amino acid composition have the same isoelectric point and hydrophobicity.

Protein Structure

• Tertiary and quaternary structures describe the 3D shape of a protein, stabilized by hydrophobic, ionic, hydrogen bonds, and sometimes disulfide bonds.
• Hydrophilic residues, like those in reflectins, form hydrogen bonds with water, which helps them stay soluble but do not drive protein folding.
• Basicity doesn’t prevent folding; many basic proteins fold well if they have hydrophobic regions.
• Secondary structure is stabilized by hydrogen bonds between the backbone carbonyl oxygen and amide hydrogen, forming α-helices and β-sheets.
• Tertiary structure is stabilized by hydrophobic interactions, hydrogen bonds, ionic bonds, van der Waals forces, and sometimes disulfide bonds between side chains.
• Quaternary structure involves multiple polypeptide subunits held together by the same interactions as tertiary structure.

Gated Channels

• It opens after a change in membrane potential → Voltage-gated.
• It opens when a molecule binds to it → Ligand-gated.
• It opens with a change in pH (H⁺ concentration) → pH-gated (subset of ligand-gated).
• It opens from physical pressure/stretch → Mechanically gated.

Miscellaneous

• Proteins with ubiquitin tags are typically targeted to the proteasome for destruction.
• A primary alcohol means the –OH group is attached to a carbon that is itself attached to only one other carbon (and two hydrogens).
• Amino acids with –OH, –SH, or –NH₂ in their side chains can act as nucleophiles in the enzyme's active site, especially if it's deprotonated.
• Amino acids with –OH, –NH₂, –SH, or an imidazole ring in their side chains can act as nucleophiles, especially in enzyme active sites.
• Schiff base formation requires a nucleophilic primary amine, which reacts with a carbonyl group (like an aldehyde) to form an imine linkage.
• Primary Amine: A primary amine has a nitrogen atom bonded to one carbon and two hydrogens (–NH₂).
• Lysine is the only amino acid with a primary amine in its side chain among the standard amino acids.
• Histones are proteins that bind DNA through electrostatic interactions between the negatively charged DNA backbone and positively charged histone.
• Ca²⁺ enters the cell through a voltage-gated channel after depolarization.
• Depolarization changes the membrane potential, opening the channel and allowing Ca²⁺ to flow into the cell down its gradient.
• Calcium enters the cell through voltage-gated channels by passive transport, moving down its concentration gradient, which does not require energy.
• The Na⁺/Ca²⁺ exchanger is not directly activated by membrane voltage; instead, it functions based on ion concentration gradients, typically removing Ca²⁺ from the cell after it accumulates; voltage changes activate channels, not exchangers.
• Accuracy requires comparison to a known gold standard; a result is only valid if it is both precise and accurate; repetition alone checks precision, not accuracy or validity.
• Precision refers to how consistent results are across repeated trials, while accuracy requires comparison to a known gold standard. A result is only valid if it is both precise and accurate. Repetition alone checks precision, not accuracy or validity.
• "Branched alkyl = L, I, V”
• SDS-PAGE: smaller proteins migrate faster.
• In SDS-PAGE, proteins that are more susceptible to degradation show fainter or fewer bands at increasing protease concentrations because they are broken down into fragments that are either too small to detect or diffuse through the gel.
• Band shift (higher molecular weight) indicates extracellular.
• No shift (same molecular weight) indicates intracellular = cytosolic, i.e., inside the cell, specifically in the cytoplasm.
• Acidic amino acids like glutamate and aspartate are charged at neutral pH and become neutral at low pH, making them less water-soluble and better able to interact with membranes.
• Western blotting commonly uses SDS-PAGE to separate proteins by size before transferring them to a membrane for antibody detection.
• In SDS-PAGE and western blots, SDS gives all proteins a negative charge, so the protein’s natural pI doesn't affect migration or detection in this context.
• Blotting techniques involve the separation of biomolecules by electrophoresis and the detection of a specific molecule using a specific probe.
• If the protein folds naturally under the given conditions, then folding is spontaneous → ΔG° is negative; if the protein stays unfolded unless helped, then folding is not spontaneous → ΔG° is positive.
• If the protein folds naturally under the given conditions, then folding is spontaneous → ΔG° is negative.
• If the protein stays unfolded unless helped, then folding is not spontaneous → ΔG° is positive.
• Western blots use probes that are not nucleic acids.
• Linear epitopes come from the primary structure and are not affected by denaturation, and conformational (folded) epitopes depend on the protein’s 3D shape and are lost when the protein is denatured by SDS.