Unit 2: Protein chemistry

Proteins are linear heteropolymers of L-α-amino acids whose biological behaviour is dictated entirely by the three-dimensional shape their sequence adopts. Anfinsen's principle (1961) — that the native fold is encoded in the amino-acid sequence under given conditions — is the governing idea this unit rests on: structure determines function, and structure itself is thermodynamically selected.

• Monomer: 20 standard amino acids sharing a common backbone (–NH–CαH(R)–CO–) and differing only in side chain R.
• Peptide bond: the C–N amide bond, partial double-bond character (~40%), planar and predominantly trans.
• Backbone freedom: rotation is restricted to two dihedral angles per residue, φ and ψ.
• Driving forces: hydrogen bonds, van der Waals contacts, electrostatics, and the hydrophobic effect, each individually weak (a few kJ mol⁻¹) but collectively decisive.
• Chirality: all residues are L-form, so backbones and secondary structures are inherently handed (e.g. right-handed α-helix).

II. Protein Function — sequence expressed as biological work

Proteins are the principal functional machinery of the cell, and every function is a consequence of a specific folded surface.

A. Categories of function

• Catalysis: enzymes accelerate reactions up to ~10¹⁷-fold; e.g. carbonic anhydrase hydrates CO₂ at near diffusion-limited rates.
• Transport and storage: haemoglobin carries O₂ via cooperative binding at four haem groups; ferritin stores iron.
• Structure: collagen (triple helix) and keratin provide mechanical strength.
• Motion: myosin and actin convert ATP hydrolysis into force.
• Signalling and regulation: receptors, kinases, and transcription factors transmit and interpret information.
• Defence: immunoglobulins recognise antigens through hypervariable loops.

B. Structure–function relationship

• Active/binding site: function localises to a small pocket where specific residues are held in precise geometry; mutating one catalytic residue can abolish activity.
• Specificity: shape and chemical complementarity (lock-and-key / induced fit) select the correct ligand.
• Allostery: binding at one site alters affinity at another, giving sigmoidal responses (haemoglobin O₂ curve).
• Conformational change: function often requires a switch between defined states (open/closed, T/R), so flexibility is itself functional.

III. Hierarchical Structure of Proteins — four organisational levels

Protein architecture is described at four nested levels, each stabilised by distinct interactions and each constraining the next.

A. Primary structure

• Definition: the covalent sequence of residues from N-terminus to C-terminus, plus disulfide (–S–S–) linkages.
• Anchor: stabilised only by covalent bonds; encodes all higher levels.
• Consequence: a single substitution (Glu→Val at position 6 of β-globin) produces sickle-cell haemoglobin.

B. Secondary structure

• Definition: local, regular backbone conformations held by main-chain hydrogen bonds.
• α-helix: 3.6 residues/turn, pitch 0.54 nm, H-bond between C=O of residue i and N–H of i+4.
• β-sheet: extended strands H-bonded laterally; parallel or antiparallel, giving a pleated surface.
• Turns and loops: β-turns reverse chain direction in four residues, often with Gly/Pro.

C. Tertiary structure

• Definition: the full three-dimensional fold of one polypeptide.
• Stabilisers: hydrophobic core packing, side-chain H-bonds, salt bridges, disulfides.
• Motifs and domains: recurring units (β-hairpin, Rossmann fold) assemble into semi-independent domains (~100–150 residues) that often fold and function alone.

D. Quaternary structure

• Definition: assembly of two or more folded subunits into a functional complex.
• Example: haemoglobin is an α₂β₂ tetramer; the interfaces mediate cooperativity.
• Interactions: same non-covalent forces as tertiary, acting between chains.

IV. Torsional angles in proteins and nucleic acids — the dihedral description of backbones

Backbone conformation is captured by rotation about single bonds, and allowed combinations are sharply limited by steric clash.

A. Torsional angles in proteins

• The three angles: per residue, φ (rotation about N–Cα), ψ (about Cα–C), and ω (about the peptide C–N bond).
• ω is fixed: planarity restricts ω to ~180° (trans) or occasionally 0° (cis, chiefly at proline).
• Free variables: only φ and ψ vary meaningfully, so a fold is essentially a list of (φ, ψ) pairs.

Backbone:  ...–N–Cα–C–N–Cα–C–...
            φ↑  ψ↑ ω↑
φ = rotation about N–Cα
ψ = rotation about Cα–C(=O)
ω = rotation about C–N (peptide bond, planar)

B. The Ramachandran plot

• Purpose: a 2-D map of φ (x) versus ψ (y) showing sterically allowed regions.
• Allowed zones: right-handed α-helix (φ≈–60°, ψ≈–45°); β-sheet (φ≈–120°, ψ≈+120°); left-handed helix (small region).
• Glycine and proline: Gly (no side chain) samples far more of the map; Pro is locked (φ≈–60°) by its ring.
• Use: disallowed points flag modelling or refinement errors in solved structures.

C. Torsional angles in nucleic acids

• Six backbone angles: α, β, γ, δ, ε, ζ define the sugar–phosphate chain (P–O5′–C5′–C4′–C3′–O3′–P).
• Glycosidic angle χ: rotation about the base–sugar bond, giving anti (common in B-DNA) or syn conformers.
• Sugar pucker: C2′-endo (B-form) versus C3′-endo (A-form/RNA) sets helix geometry.
• Contrast with proteins: nucleic-acid backbones need many angles yet occupy few discrete conformers, whereas protein backbones need only φ/ψ but explore continuous space.

V. Characterization of secondary structure using CD — chirality as a spectroscopic probe

Circular dichroism measures the differential absorption of left- and right-handed circularly polarised light, and because protein folds are chiral each secondary-structure type gives a diagnostic spectrum.

A. Principle

• Measured quantity: ΔA = A_L − A_R, reported as molar ellipticity [θ].
• Relation: [θ] ≈ 3298·Δε, converting absorbance difference to ellipticity.
• Origin of signal: the peptide bond (n→π ~222 nm, π→π ~208 and 190 nm) is the chromophore in the far-UV; its environment in a regular fold splits and shifts these transitions.

B. Diagnostic spectra

• α-helix: double negative minima at 208 nm and 222 nm plus a strong positive band near 192 nm.
• β-sheet: single broad minimum near 216–218 nm and a positive band around 195 nm.
• Random coil: strong negative band near 200 nm, little signal above 210 nm.

C. Applications and limitations

• Composition estimate: far-UV spectra are deconvoluted (e.g. by reference-set fitting) into % helix/sheet/coil.
• Folding monitor: loss of the 222 nm band tracks thermal or chemical unfolding in real time.
• Near-UV CD (250–320 nm): reports tertiary structure via aromatic side chains.
• Limitation: gives global averages, not residue-level detail; requires low-absorbance buffers and dilute, aggregate-free samples.

VI. Protein stability and folding — thermodynamics and kinetics of the native state

The native fold is only marginally stable and is reached rapidly despite an astronomically large conformational space; stability is a thermodynamic question and folding a kinetic one.

A. Protein stability

• Net stability: ΔG_folding is typically only −20 to −60 kJ mol⁻¹, a small difference between large opposing terms.
• Stabilising forces: hydrophobic burial of non-polar side chains (the dominant term), H-bonds, salt bridges, disulfides.
• Destabilising term: the large conformational entropy lost on folding opposes the process.

ΔG = ΔH − TΔS
ΔG < 0  → folded (native) state favoured
ΔG(unfolding) = −RT ln K   ;   K = [U]/[N]

where N = native, U = unfolded, K = equilibrium constant, R = gas constant, T = temperature.

B. Folding pathways and models

• Levinthal's paradox: random search of ~3¹⁰⁰ conformations would take longer than the universe's age, yet folding occurs in µs–s — so folding must be directed.
• 1. Framework/hierarchical model: secondary structures form first, then dock into tertiary structure.
• 2. Hydrophobic-collapse model: rapid burial of hydrophobic residues gives a compact molten globule, within which structure matures.
• Energy landscape: a funnel-shaped free-energy surface channels many routes downhill to the native minimum, reconciling both views.
• Intermediates: the molten globule has native-like secondary structure but a loosely packed core.

C. Denaturation and assisted folding

• Denaturants: heat, urea, guanidinium chloride, pH extremes, and detergents disrupt non-covalent forces; two-state N⇌U transitions are monitored by CD or fluorescence.
• Reversibility: Anfinsen's ribonuclease experiment showed refolding after removal of urea and β-mercaptoethanol, proving sequence encodes fold.
• Chaperones: GroEL/GroES and Hsp70 prevent aggregation and give misfolded chains further folding attempts, without altering the final structure.
• Misfolding disease: aberrant folding into β-rich amyloid fibrils underlies Alzheimer's and prion pathologies, showing that kinetic traps can be biologically catastrophic.