Structure and function of proteins and their constituent amino acids

The four classes:

• Nonpolar / hydrophobic — Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, Trp. Bury in the protein's hydrophobic core, away from water.
• Polar uncharged — Ser, Thr, Cys, Tyr, Asn, Gln. Sit on the surface; form hydrogen bonds.
• Acidic (negative at physiological pH) — Asp (D) and Glu (E); carboxyl side chains, deprotonated at pH 7.
• Basic (positive at physiological pH) — Lys (K), Arg (R), and His (H); amino/guanidinium/imidazole side chains.

A useful shortcut: the aromatic amino acids (Phe, Trp, Tyr) absorb UV light at 280 nm, which is how protein concentration is measured.

• Glycine — R group is just H, so it is the only achiral amino acid and the smallest; its flexibility lets it fit tight turns.
• Proline — its side chain loops back to the backbone nitrogen (a secondary amine), making it rigid; it disrupts α-helices and is common at turns and kinks.
• Cysteine — its thiol (–SH) can oxidize with another cysteine to form a covalent disulfide bond, a key stabilizer of tertiary/quaternary structure (and the bond that reducing agents and SDS break).
• Histidine — its imidazole side chain has a pKa near 6, so it can pick up or give off a proton at physiological pH; this makes it both a biological buffer and a frequent acid–base catalyst in enzyme active sites.

Each amino acid has at least two pKa values — the α-carboxyl (~2) and the α-amino (~9–10) — plus a third for any ionizable side chain. As pH rises, groups deprotonate in order of pKa. At a pH below the pI the molecule is net positive (protonated); at a pH above the pI it is net negative. The pI is the average of the two pKa values that flank the neutral (zwitterionic) form: for an amino acid with no ionizable side chain, that's the average of the α-COOH and α-NH₃⁺ pKa values; for an acidic amino acid it's the average of the two lowest pKa values; for a basic one, the average of the two highest.

Primary structure is the order of residues from N- to C-terminus. A single substitution can be catastrophic — sickle-cell anemia is one Glu→Val change in β-globin that creates a hydrophobic patch and makes hemoglobin polymerize. Primary structure is determined experimentally by sequencing (Edman degradation, mass spectrometry).

In the α-helix, the backbone coils and each carbonyl O hydrogen-bonds to the amide H four residues ahead (i → i+4); side chains point outward. Proline kinks and glycine destabilizes helices. In the β-pleated sheet, extended strands lie side by side and hydrogen-bond across to one another, running parallel or antiparallel. The defining point: secondary structure is backbone hydrogen bonding — independent of which side chains are present.

Tertiary structure is where side chains do the work. The dominant driving force is the hydrophobic effect: nonpolar residues cluster away from water, which is entropically favorable. Salt bridges (between acidic and basic side chains), hydrogen bonds, and disulfide bonds then lock the fold in place. Tertiary structure is what gives an enzyme its active-site geometry.

Quaternary structure is held by the same interactions as tertiary, but between subunits. The classic example is hemoglobin (four subunits), whose subunit cooperation produces sigmoidal O₂ binding. Multi-subunit assembly enables cooperativity and allosteric regulation.

Folding is driven by the sequence (Anfinsen's classic experiment: denatured ribonuclease refolds on its own once the denaturant is removed), but in the crowded cell chaperones (e.g., heat-shock proteins, chaperonins) prevent misfolding and aggregation. Denaturants — heat, extreme pH, urea, detergents like SDS, and reducing agents (which break disulfides) — disrupt the higher-order structure but do not break peptide bonds. Misfolding underlies diseases like the prion encephalopathies and amyloidoses.

Ubiquitination is a post-translational modification: enzymes covalently link ubiquitin to a lysine of the target, and a polyubiquitin chain marks it for destruction. The barrel-shaped proteasome then recognizes the tag, unfolds the protein, and chops it into peptides — the cell's main route for regulated, selective protein turnover. This is how short-lived regulatory proteins are kept in check: it is the mechanism behind the fall in cyclin levels that drives the cell cycle forward and behind the rapid turnover of damage-sensing proteins like p53.

The lock-and-key model says the substrate fits a pre-shaped active site; the induced-fit model (Koshland) — the better description — says binding induces a conformational change that brings catalytic groups into position. Helpers: cofactors are inorganic (e.g., Zn²⁺, Mg²⁺, Fe²⁺); coenzymes are small organic molecules, many derived from vitamins (NAD⁺ from niacin, FAD from riboflavin, coenzyme A from pantothenate). An enzyme without its cofactor is an inactive apoenzyme; with it, an active holoenzyme. A tightly/covalently bound cofactor is a prosthetic group (e.g., heme).

The Michaelis–Menten equation, v = Vmax·[S] / (Km + [S]), gives a hyperbolic curve: rate is roughly first-order in substrate at low [S] and plateaus (zero-order) at Vmax when every active site is occupied. Km equals the [S] at which v = ½Vmax; a small Km means the enzyme reaches half-speed at low substrate, i.e., binds tightly. kcat (turnover number) is Vmax/[E]ₜₒₜₐₗ, and kcat/Km measures catalytic efficiency. The Lineweaver–Burk double-reciprocal plot linearizes the data (y-intercept = 1/Vmax, x-intercept = −1/Km), which is why inhibitor questions are usually drawn that way. Cooperative (allosteric) enzymes break the rule with a sigmoidal curve.

The four cases:

The intuition: a competitive inhibitor competes for the active site, so flooding with substrate outcompetes it (Vmax recoverable, apparent Km worsens). A noncompetitive inhibitor binds elsewhere and takes enzyme out of play no matter how much substrate you add (Vmax falls; affinity, hence Km, unchanged). An uncompetitive inhibitor binds only after substrate binds, locking the ES complex (both Km and Vmax fall).

Allosteric enzymes have regulatory sites distinct from the active site; binding of an effector shifts the enzyme between high- and low-activity states, producing a sigmoidal (cooperative) rate curve. In feedback (end-product) inhibition, a pathway's final product inhibits an early enzyme, preventing overproduction. Covalent modification — most often phosphorylation by kinases (reversed by phosphatases) — switches enzymes on or off. Zymogens (proenzymes like pepsinogen, trypsinogen, and the clotting factors) are made inactive and switched on by proteolytic cleavage, so destructive enzymes aren't active until and where they're needed. Cooperativity (as in hemoglobin) means binding at one site changes affinity at the others.

This is the top-level split that sits above the four kinetic classes. Reversible inhibitors associate and dissociate freely, so the enzyme regains activity when the inhibitor is diluted out or outcompeted by substrate, and they shift Km/Vmax in the patterns of the kinetics table. An irreversible inhibitor instead reacts covalently — usually with a catalytic residue in or near the active site (e.g., a serine or cysteine) — so that one enzyme molecule is permanently knocked out. Removing or washing out the inhibitor does not restore activity; because the enzyme is destroyed rather than merely blocked, the only way for a cell to recover function is to synthesize new enzyme. Classic examples: organophosphates and nerve agents covalently modifying acetylcholinesterase, penicillin acylating a bacterial transpeptidase, and aspirin acetylating cyclooxygenase (COX).

Hemoglobin's four subunits cooperate: binding one O₂ raises affinity for the next, giving the S-shaped curve that lets it load O₂ in the lungs and unload it in tissues. Its affinity is tuned by the environment — the Bohr effect: lower pH, higher CO₂, higher temperature, and higher 2,3-BPG all right-shift the curve (lower affinity), dumping O₂ where metabolism is high. Myoglobin has one heme, no cooperativity, and a hyperbolic curve sitting to the left (higher affinity) — perfect for grabbing and holding O₂ in muscle.

In SDS-PAGE, the detergent SDS denatures proteins and coats them with uniform negative charge, so migration depends only on molecular weight — smaller proteins travel farther. Native PAGE omits SDS, so folded proteins separate by a mix of size, shape, and charge. Isoelectric focusing runs proteins through a pH gradient; each protein migrates until it reaches the pH equal to its pI, where its net charge is zero and it stops. Running IEF and then SDS-PAGE perpendicular to it is 2-D gel electrophoresis, which resolves complex mixtures.

In size-exclusion (gel-filtration) chromatography, large proteins can't enter the porous beads and elute first, while small ones are delayed (counterintuitive — big comes out fast). Ion-exchange columns carry a charge and retain oppositely charged proteins, which are then released by raising salt or changing pH. Affinity chromatography uses a column-bound ligand (an antibody, a substrate analog, or a metal for a His-tag) to capture only the protein of interest — the most specific and most powerful purification step.

A primary antibody binds the target antigen; a secondary antibody carrying an attached enzyme then binds, and adding the enzyme's substrate produces a colored product. The absorbance read by a plate reader is proportional to the amount of antigen, which makes ELISA the go-to quantitative answer when a passage asks how much protein (or hormone, or antibody) is in a sample. The classic format is the sandwich ELISA: a capture antibody coats the well, antigen binds, and a detection antibody completes the sandwich.

SDS denatures a protein and masks its charge, but it cannot cleave the covalent disulfide bonds that link cysteines across separate chains. So under non-reducing conditions, disulfide-bonded subunits migrate together as a single high-molecular-weight band — SDS-PAGE is "size only" for what SDS can pull apart, not for covalently tethered subunits. Adding a reducing agent breaks the disulfides; now each chain runs as its own band at its true size. The textbook AAMC example is an antibody (~150 kDa) that appears as one band non-reduced but resolves into separate heavy and light chains only when a reducing agent is added.