Amino Acids & Proteins

Amino Acids & Proteins

Part 1 of 4 — The 20 Amino Acids: Structure & Classification

Anatomy of an Amino Acid

Every proteinogenic amino acid shares the same backbone: a central $\alpha$-carbon bonded to an amino group ($-NH_{2}$), a carboxyl group ($-COOH$), a hydrogen, and a variable side chain (R group). At physiological pH, the backbone exists as a zwitterion: $-NH_{3}^{+}$ and $-COO^{-}$ simultaneously, giving a net backbone charge of zero.

All 20 standard amino acids are L-stereoisomers with (S) absolute configuration — except cysteine, which is (R) (the sulfur raises the side chain's priority), and glycine, which is achiral (two hydrogens on the $\alpha$-carbon).

Classification by Side Chain — Know This Cold

Tyrosine caution: its phenol -OH makes it polar-ish, but the MCAT usually groups it with the aromatics; it is a common phosphorylation site (along with Ser and Thr).

The Special Cases the MCAT Loves

• Glycine (G): side chain = H. Smallest, most flexible, achiral. Fits in tight turns; every third residue of collagen is Gly.
• Proline (P): side chain cyclizes back onto the backbone nitrogen (a secondary amine — technically an imino acid). Rigid; introduces kinks; helix breaker.
• Cysteine (C): thiol ($-SH$) side chain; two cysteines oxidize to form a covalent disulfide bond (cystine).
• Histidine (H): imidazole side chain with pKa about 6 — the only side chain that titrates near physiological pH, making it a superb acid-base catalyst in active sites (e.g., serine proteases, carbonic anhydrase).
• Methionine (M): thioether sulfur (no disulfides!); as N-formylmethionine (bacteria) or Met (eukaryotes) it initiates translation.

Hydrophobicity Predicts Location

In a water-soluble globular protein, nonpolar residues (V, L, I, F, M) cluster in the interior; charged/polar residues face the aqueous exterior. In a transmembrane segment, the pattern inverts: hydrophobic residues face the lipid bilayer. Passage questions often give you a mutation (e.g., Ile to Asp in a membrane-spanning helix) and ask you to predict destabilization.

Amino Acid Structure & Classification 🎯

Key Takeaways — Part 1

• Backbone = zwitterion at pH 7; all are L (S-configuration) except Cys (R) and achiral Gly
• Classify instantly: nonpolar (G, A, V, L, I, P, M), aromatic (F, W, Y), polar (S, T, C, N, Q), acidic (D, E), basic (K, R, H)
• Special residues: Gly = flexible, Pro = rigid helix breaker, Cys = disulfides, His = pKa ~6 catalyst, Met = start codon
• Hydrophobic in = soluble protein core; hydrophobic out = transmembrane segment
• Trp/Tyr absorb at 280 nm (protein quantification)

Worked Examples — Amino Acid Identification

Question: A cytosolic enzyme's hydrophobic core contains a valine. Which substitution is LEAST likely to disrupt folding: Val to Ile, Val to Lys, or Val to Asp?

Solution:

1. Val is small and nonpolar, buried in the hydrophobic core.
2. Ile is also nonpolar and nearly the same size — a conservative substitution. Minimal disruption.
3. Lys (+) and Asp (−) are charged; burying a charge in the nonpolar core carries a large energetic penalty and can misfold the protein.

Answer: Val to Ile.

MCAT Strategy: "Conservative" = similar chemistry (nonpolar for nonpolar, charge for like charge). The exam tests whether you match side-chain chemistry to environment, not memorized structures.

Question: A protein is inactivated by a reagent that alkylates free thiol groups. Site-directed mutagenesis of one residue to serine abolishes sensitivity to the reagent but preserves most activity. What was the original residue?

Solution:

1. Free thiol ($-SH$) means cysteine.
2. Serine is cysteine's oxygen analog ($-OH$ in place of $-SH$): similar size, no thiol, so the alkylating agent no longer reacts.
3. Retained activity indicates the thiol was not strictly catalytic but was the modification target.

MCAT Strategy: Cys-to-Ser swaps are the classic experiment for testing whether a specific thiol matters. Met also contains sulfur but as a thioether — it does not react like a free thiol and cannot form disulfides.

Question: Collagen's triple helix requires glycine at every third position (Gly-X-Y, with X often Pro). A mutation replaces one such glycine with arginine. Why is this so destabilizing?

Solution:

1. In the tightly wound triple helix, every third residue packs at the crowded central axis where the three chains meet.
2. Only glycine — side chain = H — is small enough to occupy that position.
3. Arginine's long, bulky, charged side chain cannot fit, disrupting the helix (the basis of diseases such as osteogenesis imperfecta).

MCAT Strategy: When a question says a mutation of Gly is uniquely damaging, think steric constraint: Gly marks positions where there is literally no room for a side chain.

Amino Acids & Proteins

Part 2 of 4 — Acid-Base Chemistry, pKa & Isoelectric Point

Henderson-Hasselbalch: The One Equation You Must Own

$pH = pK_a + \log\frac{[A^-]}{[HA]}$

• When $pH = pK_a$: 50/50 mix of protonated and deprotonated forms (maximum buffering).
• When $pH > pK_a$ (by ~2 units): the group is essentially deprotonated.
• When $pH < pK_a$ (by ~2 units): the group is essentially protonated.

Benchmark pKa Values (approximate — learn the pattern, not decimals)

Isoelectric Point (pI)

The pI is the pH at which the molecule's net charge is zero. For a simple amino acid:

$pI = \frac{pK_{a1} + pK_{a2}}{2}$

...where the two pKa values are those flanking the neutral (zwitterionic) species:

• Neutral side chain (e.g., Gly, Ala): average the two backbone pKa values. $pI = (2.3 + 9.6)/2 \approx 6$.
• Acidic (Asp, Glu): average the two lowest pKa values (the two carboxyls). pI ~3.
• Basic (Lys, Arg, His): average the two highest pKa values (the two amines). pI ~10 (Lys), ~11 (Arg), ~7.6 (His).

Titration Curves

A titration curve for an amino acid shows one buffering plateau per ionizable group (flat region centered at each pKa) and steep rises between them. The pI sits on a steep region for neutral amino acids — halfway between the two pKa values — NOT at a plateau. Amino acids with ionizable side chains show three plateaus.

Why This Matters Experimentally

• Electrophoresis / isoelectric focusing: at pH > pI a protein is net negative (migrates to the anode); at pH < pI it is net positive; at pH = pI it does not migrate and is least soluble (proteins often precipitate at their pI).
• Ion-exchange chromatography: choose the column based on the protein's charge at the working pH (next-level detail in Part 4).

pKa, Buffers & pI 🎯

Key Takeaways — Part 2

• Henderson-Hasselbalch: each unit of pH above pKa multiplies the deprotonated:protonated ratio by 10
• pI = average of the two pKa values flanking the neutral species: two lowest for acidic AAs, two highest for basic AAs
• pH > pI: net negative, runs to anode; pH < pI: net positive, runs to cathode; pH = pI: no migration, minimum solubility
• His (pKa ~6) is the physiological buffer residue; Asp/Glu are always negative and Lys/Arg always positive at pH 7
• Titration curves: one plateau per ionizable group; buffering only near pKa

Worked Examples — Charge Calculations

Question: Find the net charge of the tetrapeptide Glu-His-Lys-Ala at pH 7.4. (pKa values: Glu side chain 4.3; His side chain 6.0; Lys side chain 10.5; N-terminus 9.0; C-terminus 3.1)

Solution:

1. N-terminus (pKa 9.0): pH < pKa, protonated → +1
2. C-terminus (pKa 3.1): pH > pKa, deprotonated → −1
3. Glu side chain (4.3): pH > pKa → −1
4. His side chain (6.0): pH > pKa, mostly deprotonated → 0 (approximately)
5. Lys side chain (10.5): pH < pKa → +1
6. Ala: nonpolar → 0

Net charge ≈ +1 − 1 − 1 + 0 + 1 = 0

MCAT Strategy: Only termini and ionizable side chains count. Internal residues in a peptide contribute no backbone charges — their carboxyls and amines are tied up in peptide bonds.

Question: Protein A has pI 5.0; protein B has pI 9.0. At what pH would electrophoresis send them in opposite directions?

Solution:

1. Pick a pH between the two pI values, e.g., pH 7.
2. Protein A: pH 7 > pI 5.0 → net negative → migrates to anode.
3. Protein B: pH 7 < pI 9.0 → net positive → migrates to cathode.

MCAT Strategy: "Between the pIs" is the universal answer for opposite-direction separation. At a pH equal to one protein's pI, that protein simply stays put.

Question: A titration curve of an unknown amino acid shows buffering plateaus at pH 2.2, 6.8, and 9.1. Which amino acid is it likely to be, and what is its pI?

Solution:

1. Three plateaus = three ionizable groups = an amino acid with an ionizable side chain.
2. The middle plateau near 6.8 points to an imidazole — histidine is the only standard amino acid with a side-chain pKa in that range. The unknown is histidine (tabulated pKa values ~1.8, 6.0, 9.2; measured values vary with conditions).
3. His is basic, so pI = average of the two HIGHEST pKa values: $(6.8 + 9.1)/2 \approx 7.95$.

MCAT Strategy: A mid-range plateau (pKa 5-8.5) narrows the identity fast: ~6 is His, ~8 is Cys. Then apply the flanking-pKa rule for pI.

Amino Acids & Proteins

Part 3 of 4 — Protein Structure, Folding & Denaturation

The Peptide Bond

Peptide bonds form by condensation (dehydration) between one residue's carboxyl and the next residue's amino group, releasing water. Key properties:

• Partial double-bond character from resonance → the bond is planar and rigid; rotation occurs only around the flanking $\phi$ and $\psi$ backbone bonds.
• Almost always trans configuration (side chains on opposite sides) to avoid steric clash; proline is the notable exception that tolerates cis.
• Hydrolysis is thermodynamically favorable but kinetically slow — proteases (and strong acid + heat) provide the catalysis.

The Four Levels of Structure

What Actually Drives Folding: The Hydrophobic Effect

Burying nonpolar side chains releases ordered water molecules from around them, increasing the entropy of the solvent. This entropy gain is the dominant thermodynamic driver of folding — more important than any individual H-bond. Folding is spontaneous for many small proteins (Anfinsen's ribonuclease experiment: sequence alone encodes the native fold), but large proteins in the crowded cell often need chaperones (e.g., heat shock proteins) to prevent aggregation, not to dictate the fold.

Secondary Structure Details

• $\alpha$-helix: right-handed; C=O of residue $i$ H-bonds to N-H of residue $i+4$; side chains point outward; ~3.6 residues per turn. Breakers: Pro (no N-H, rigid) and Gly (too flexible).
• $\beta$-sheet: extended strands H-bonded side by side; antiparallel sheets have straighter, stronger H-bond geometry than parallel; side chains alternate above/below the sheet plane.

Denaturation

Denaturation destroys 2°-4° structure while leaving the primary sequence intact:

Misfolding has clinical stakes: aggregated $\beta$-sheet-rich conformers underlie amyloid diseases and prion pathology.

Protein Structure & Folding 🎯

Key Takeaways — Part 3

• Peptide bond: planar (resonance), trans-preferring, kinetically stable; formed by condensation, broken by hydrolysis
• 1° sequence → 2° backbone H-bonding ($\alpha$-helix i to i+4; $\beta$-sheets) → 3° side-chain interactions → 4° multi-subunit assembly
• Hydrophobic effect (solvent entropy) drives folding; sequence encodes the fold (Anfinsen); chaperones prevent aggregation
• Match the denaturant to its target: reducing agents = disulfides only; SDS = hydrophobic; pH = ionic/H-bonds
• Cooperativity (hemoglobin) = quaternary phenomenon

• Low-yield extras: in vivo, correct disulfide pairing is often aided by the ER enzyme protein disulfide isomerase (PDI), which reshuffles mispaired disulfides

Worked Examples — Structure & Denaturation Logic

Question: A mutation changes a surface glutamate to lysine, breaking a salt bridge with an arginine on a neighboring subunit and causing the tetramer to dissociate into monomers. Each monomer remains folded and soluble. Which structural level is disrupted?

Solution:

1. The salt bridge is BETWEEN subunits — an intersubunit contact.
2. Monomers still fold (tertiary intact); only the assembly falls apart.
3. Quaternary structure is disrupted.

MCAT Strategy: Ask "within one chain or between chains?" The same chemical interaction (salt bridge, H-bond) can be tertiary or quaternary depending on where it acts.

Question: An enzyme retains activity in 8 M urea but loses activity when $\beta$-mercaptoethanol is added along with the urea. Activity does not return on dilution. What does this suggest about the enzyme?

Solution:

1. Stability in urea alone → the fold is unusually robust to loss of noncovalent interactions, implying covalent crosslinks hold it together.
2. Adding a reducing agent destroys activity → disulfide bonds were the critical stabilizers.
3. No recovery after dilution → the reduced protein misfolds or aggregates instead of re-forming its native disulfide pairs; the disulfides likely needed to form in a specific order.

MCAT Strategy: Urea vs. reducing agent is the classic experimental dissection: urea = noncovalent, $\beta$-ME/DTT = covalent disulfides.

Question: Two peptide segments are compared: segment 1 is Ala-Leu-Glu-Lys-Met-Ala; segment 2 is Pro-Gly-Pro-Ser-Gly-Pro. Which is more likely to form an $\alpha$-helix?

Solution:

1. Segment 1 contains good helix formers (Ala, Leu, Met, Glu, Lys) and no breakers.
2. Segment 2 is loaded with Pro (rigid, no N-H for helix H-bonding) and Gly (too flexible) — a helix-hostile composition; this Pro-Gly-rich pattern is actually collagen-like.
3. Segment 1 forms the helix.

MCAT Strategy: You only need two facts: Pro and Gly disfavor $\alpha$-helices, and Ala is the prototypical helix former.

Amino Acids & Proteins

Part 4 of 4 — Protein Purification, Analysis & MCAT Integration

The MCAT tests proteins mostly through experimental passages. You must know what each technique separates by, and how to read its output.

Separation Techniques

SDS-PAGE with vs. without $\beta$-mercaptoethanol: reducing conditions split disulfide-linked subunits. A protein running at 150 kDa nonreduced but 50 kDa reduced is a disulfide-linked trimer (or has interchain disulfides).

Quantification & Identification

• UV 280 nm: Trp/Tyr absorbance; fast, nondestructive.
• Bradford assay: colorimetric total-protein assay read on a spectrophotometer (Beer's law: $A = \epsilon l c$).
• Western blot: SDS-PAGE → transfer → antibody detection of one specific protein.
• ELISA: antibody-based detection/quantification of a specific protein in a sample, done in plate wells with no gel step.

• The BCA assay is a second colorimetric total-protein assay, read the same way as the Bradford. A sandwich ELISA uses two antibodies: a capture antibody fixed to the plate and a labelled detection antibody.

• Edman degradation: removes and identifies one residue at a time from the N-terminus; fails past ~50 residues and on blocked N-termini.
• Mass spectrometry: measures mass-to-charge; identifies proteins from peptide fragment masses.
• X-ray crystallography / cryo-EM / NMR: full 3D structure (NMR for small proteins in solution).

Activity vs. Purity Bookkeeping

Purification tables track total protein (mg), total activity (units), and specific activity (units/mg). A good purification step raises specific activity (purer enzyme) even as total activity drops slightly (some loss). Fold purification = specific activity after / specific activity before; yield = total activity after / initial total activity.

Purification & Analysis 🎯

Key Takeaways — Part 4

• Size exclusion: BIG elutes first. SDS-PAGE: SMALL runs farthest. Don't mix these up.
• Ion exchange: beads bind opposite charge; protein charge set by pH vs. pI; elute with salt
• Reducing vs. nonreducing SDS-PAGE reveals disulfide-linked subunit composition
• Edman = N-terminal sequencing (needs free N-terminus, short peptides); mass spec = identification by fragment mass
• Specific activity (units/mg) is the purity metric; fold purification compares specific activities

• Low-yield extras: the BCA assay is a second colorimetric total-protein assay; a sandwich ELISA pairs a plate-bound capture antibody with a labelled detection antibody

Worked Examples — Experimental Integration

Question: Your target protein is 25 kDa with pI 4.5. Major contaminants are 25 kDa with pI 8.0, and 150 kDa with pI 4.5. Which two techniques, in either order, purify the target?

Solution:

1. Target vs. contaminant 1: same size, different pI → separate by ion exchange (at pH 7, target is negative, contaminant 1 positive; an anion exchanger binds only the target).
2. Target vs. contaminant 2: same pI, different size → separate by size exclusion (150 kDa elutes first) or SDS-free sizing method.
3. Neither technique alone resolves both contaminants; the pair does.

MCAT Strategy: Match each contaminant to the property that differs. If two proteins share a property, that technique cannot separate them.

Question: Cells are treated with a kinase inhibitor. Western blots of lysates are probed with (a) an antibody against total protein X and (b) an antibody specific for phospho-X. Band (a) is unchanged by treatment; band (b) disappears. Conclusion?

Solution:

1. Total X unchanged → the inhibitor does not affect X's expression or stability.
2. Phospho-X lost → X is no longer phosphorylated.
3. Conclusion: X is a substrate (direct or downstream) of the inhibited kinase; the inhibitor blocks X's phosphorylation, not its production.

MCAT Strategy: Phospho-specific antibodies report modification state; always compare against the total-protein control before concluding anything about expression.

Question: A pentapeptide gives these data: Edman degradation releases Met first. Trypsin (cleaves after Lys/Arg) yields two fragments: Met-Ala-Lys and Gly-Phe. Chymotrypsin (cleaves after aromatic residues) makes no internal cut. What is the sequence?

Solution:

1. Edman: N-terminus is Met.
2. Trypsin cuts after Lys → the fragment Met-Ala-Lys must be N-terminal; Gly-Phe follows.
3. Sequence: Met-Ala-Lys-Gly-Phe. Chymotrypsin is consistent: the only aromatic (Phe) is already the C-terminus, so no internal cut.

MCAT Strategy: Anchor the N-terminus with Edman data, then order fragments so cleavage-site residues (Lys/Arg for trypsin; Phe/Trp/Tyr for chymotrypsin) sit at fragment C-termini.