pH-abulous Foundations
At the pKa of an ionizable group, the molecule exists in this ratio of protonated to deprotonated forms
What is 1:1?
Explanation:
- When pH = pKa, [HA] = [A-]
- Using Henderson-Hasselbach: pH = pKa + log[A-]/[HA] -> 0=log[A-]/[HA] -> [A-]/[HA] = 1 so [A]=[HA]. Molecule is 50% protonated and 50% deprotonated
This amino acid has a single hydrogen atom as its side chain, making it the smallest amino acid.
What is glycine?
These are the 6 common protein tertiary structure motifs.
What is a coiled coil, helix bundle, beta-alpha-beta unit, beta barrel, greek key, and hairpin?
This technique relies on the emission of light from a donor fluorophore which can excite a nearby acceptor fluorophore
What is FRET?
These types of substrates generate coloured products and can be measured to monitor an enzyme’s activity.
What are chromogenic substrates?
At physiological pH, amino acids exist as zwitterions. This charge-separated state is stabilized not only by internal electrostatics but primarily through this process involving the surrounding water molecules.
What is solvation with water, through ion–dipole interactions between the charged groups and water’s dipoles?
Draw out the amino acid that starts every polypeptide during translation.
What is methionine?
There are this many peptide bonds in the following molecule: A–G–S–L–H–V–D–K–T–F. Additionally, this is also the identity of the amino acids.
What is...
A = Alanine; G = Glycine; S = Serine; L = Leucine; H = Histidine; V = Valine; D = Aspartic acid; K = Lysine; T = Threonine; F = Phenylalanine
10 amino acids, 9 peptide bonds.
Tip: Count the number of amino acids in your peptide (look at the backbone or the chemical formula). Subtract 1 → that’s the number of peptide bonds.
This ~thing~ forms covalent bonds between functional groups within a molecule (intramolecular) or between molecules (intermolecular). What reactive side chains are targeted specifically?
What is chemical crosslinking (i.e. formaldehyde)?
Reactive side chains of amino acids are targeted specifically:
Primary amines
Carboxyls
Carbonyls
Sulfhydryls
These are the 7 classes by which enzymes can be classified.
What are oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, and translocases?
Despite being 10–100× stronger than non-covalent interactions, covalent bonds don’t dictate protein folding because of this key property of non-covalent forces.
What is their reversibility and dynamic nature, which allows macromolecules to sample conformations and stabilize the lowest-energy structure through multiple weak interactions?
The side chain of Histidine has a pKa ≈ 6.0. At pH 4.0, what percentage of the side chain is protonated?
What is about 99% protonated?
A researcher analyzes the amino acid sequence of a 15-residue polypeptide:
Sequence:
M–L–V–I–F–A–L–V–G–L–I–F–V–L–A–G–S–T–K–E–Q–R–N–D–S–H–Y–P–C–G
Based on the properties of the amino acids, predict whether this polypeptide is most likely:
A) Soluble in the cytosol
B) Found within a lipid droplet
C) Part of a transmembrane protein
Explain your reasoning.
Look at hydrophobicity of the polypeptide
Residues 1–18: M–L–V–I–F–A–L–V–G–L–I–F–V–L–A–G–S–T are mostly hydrophobic (M, L, V, I, F, A, G) (transmembrane domain). Residues 19-30 are mostly polar/charged (extracellular/cytosolic portion)
:. Transmembrane protein
A student prepares a 0.67 cm path length cuvette containing a protein solution with an unknown concentration. The protein has a molar extinction coefficient (ε) of 42,000 M⁻¹·cm⁻¹ at 280 nm. What is the protein concentration?
A Lineweaver-Burk plot shows three lines: All intersect at the y-axis, but have different slopes and different x-intercepts. From this pattern, the type of inhibition observed is:
What is competitive inhibition?
In competitive inhibition, Vmax stays the same (y-intercept constant), but Km increases (slope increases).
That’s why the lines intersect at the y-axis on the LB plot.
The tripeptide Asp–His–Gly has pKa values 2.0, 3.9, 6.0, and 9.0. The pI occurs between the deprotonation of these two groups.
What are the side-chain (imidazole) of histidine and the α-amino terminus?
Which one of the following “polypeptide names” is made up only of valid amino acid 1-letter codes? What are the amino acids?
BAGEL 🍩
MAGIC ✨
PIZZA 🍕
QUEEN 👑
BAGEL -> not valid because of B (Alanine, Glycine, Glutamic acid, leucine)
MAGIC -> correct (methionine, alanine, glycine, isoleucine, cysteine)
PIZZA -> Z not valid (proline, isoleucine, alanine)
QUEEN -> U not valid (glutamine, glutamic acid, asparagine)
A patient with progressive cognitive decline is found to have a single amino acid substitution in a neuronal protein: lysine is replaced by leucine at a residue located deep within the protein core. The protein is later observed to accumulate in the brain as insoluble aggregates. This is the proposed biochemical pathway by the diagnosing doctor.
What is a destabilizing mutation that disrupts tertiary structure (positive to nonpolar), leading to protein misfolding and aggregation as seen in diseases like Creutzfeldt–Jakob disease or Alzheimer's disease?
Name the 4 types of protein chromatographies and how they purify proteins.
Size or shape: size-exclusion/gel filtration chromatography
Charge: ion exhcnage chromatography
Binding interactions: affinity chromatography
Hydrophobicity: RP-HPLC (Reverse Phase High pressure Liquid Chromatography)
A researcher measures the initial velocity (V0) of an enzyme at different substrate concentrations ([S]) and plots the Lineweaver-Burk double reciprocal. The resulting line has a y-intercept of 0.2 min/µM and a slope of 1.5 µM·min. Calculate Vmax and Km.
Vmax = 1/y = 1/0.2 = 5 uM/min
Km = slope x Vmax = 1.5 x 5 = 7.5 uM
Although hydrophobic interactions are often described as “nonpolar molecules sticking together,” the true energetic driving force comes from changes in this, rather than direct solute-solute attraction.
What is the entropy of water during solvation and release from structured cages?
Name all 5 non-covalent interactions that can form within a polypeptide, the groups this occurs with, and the function.
What are....
Hydrogen bonds:
Between hydroxyl, carboxyl, thiol, amino groups -> protein solubility
Between amino acid side chains or backbone within a proteins structure
Hydrophobic interactions
Between aliphatic and hydrophobic side chains
Ionic interactions
Important for ligand, cofactor and/or substrate binding in enzymes
Salt bridges
Between + and - charged amino acids
A polypeptide has an unknown sequence. A student performs the following experiments:
Cleave with trypsin (cuts after K & R) and sequence by Edman degradation reveals fragments:
T1: GASMALIK
T2: EGAAYHDFEPIDPR
T3: DCVHSD
T4: YLIACGPMTK
Cleave with CNBr (cleaves after M) and sequence by Edman degradation reveals fragments:
C1: EGAAYHDFEPIDPRGASM
C2: TKDCVHSD
C3: ALIKYLIACGPM
Can you work out the sequence?
What is EGAAYHDFEPIDPRGASMALIKYLIACGPMTKDCVHSD?
A student is purifying a 55 kDa enzyme from E. coli. After affinity chromatography, the student runs an SDS-PAGE gel and observes the following:
Lane 1 (Marker): standard protein ladder
Lane 2 (Crude lysate): many bands
Lane 3 (Elution fraction): one strong band at ~55 kDa and a faint band at ~30 kDa
Lane 4 (Wash fraction): faint smear of proteins
The student then performs a Western blot using an antibody specific for the enzyme and observes a single band at 55 kDa in lane 3.
Finally, they perform mass spectrometry on the elution fraction and detect two major peptides:
One corresponding to the target enzyme (55 kDa)
One corresponding to a contaminant (30 kDa), identified as a common E. coli chaperone protein.
Q1. Based on the SDS-PAGE and immunoblot, how pure is the enzyme preparation? Justify your reasoning.
Q2. Why might the 30 kDa contaminant have co-purified during affinity chromatography?
Q3. Propose one additional purification step that could help remove the contaminant and explain why it would be effective.
Q1:
SDS-PAGE shows two bands, so not 100 % pure.
Immunoblot detects only 55 kDa band, meaning the contaminant isn’t the target.
The prep is enriched but not completely pure.
Q2:
The contaminant may bind nonspecifically to the resin, or form a complex with the target protein, co-eluting in the same fraction.
Q3:
Gel filtration (size-exclusion chromatography) would separate 55 kDa from 30 kDa based on size.
Alternatively, ion exchange could separate based on charge differences.
A researcher measures the Michaelis constant (KmKm) of three enzymes (A, B, C) with two different substrates (X and Y). The results are:
Enzyme A: Km for X = 2 uM ; Km for Y = 10 uM
Enzyme B: Km for X = 5 uM ; Km for Y = 5 uM
Enzyme C: Km for X = 12 uM ; Km for Y = 2 uM
Which enzyme shows the highest specificity for substrate X, and which shows no substrate preference? Explain your reasoning.
Highest specificity for X: Enzyme A → low Km for X (2 µM) vs high Km for Y (10 µM)
No substrate preference: Enzyme B → Km is the same for X and Y (5 µM), so it binds both equally well
Explanation / Notes:
Lower Km → higher affinity
Comparing Km for multiple substrates allows assessment of specificity.
Enzyme C prefers Y (low Km for Y, high for X).