Biochemistry I: Protein Structure

Primary structure

Linear amino-acid sequence joined by peptide bonds.

Secondary structure

Local backbone conformations such as α-helices, β-sheets, turns and PPII.

Tertiary structure

Overall three-dimensional arrangement of one polypeptide chain.

Quaternary structure

Arrangement and interactions of multiple polypeptide subunits.

Why is the peptide bond planar?

Resonance gives the C–N peptide bond partial double-bond character, restricting rotation.

Trans peptide bond

Adjacent Cα atoms lie on opposite sides; ω ≈ 180°; most common conformation.

Cis peptide bond

Adjacent Cα atoms lie on the same side; more common before proline than other residues.

Peptidyl-prolyl cis–trans isomerase

Enzyme that catalyzes interconversion of cis and trans peptide bonds involving proline.

Phi (φ) angle

Backbone dihedral angle around the N–Cα bond.

Psi (ψ) angle

Backbone dihedral angle around the Cα–carbonyl C bond.

Omega (ω) angle

Dihedral angle around the peptide C–N bond; usually about 180° in trans peptides.

Steric clashes

Unfavorable overlap of atomic van der Waals volumes that restricts φ and ψ angles.

Ramachandran plot

Plot of φ on the x-axis versus ψ on the y-axis showing allowed backbone conformations.

Why does glycine have more allowed φ/ψ angles?

Its side chain is H, minimizing steric hindrance and increasing flexibility.

Why is proline conformationally restricted?

Its cyclic side chain connects to backbone nitrogen and restricts φ rotation.

α-helix handedness

Common protein α-helix is right-handed.

α-helix φ and ψ angles

Approximately φ = −57° and ψ = −47°.

α-helix residues per turn

3.6 amino-acid residues per turn.

α-helix pitch and rise

Pitch ≈ 5.4 Å per turn; rise ≈ 1.5 Å per residue.

α-helix hydrogen bonding

Backbone C=O of residue i hydrogen-bonds to N–H of residue i+4.

α-helix side-chain orientation

Side chains project outward from the tightly packed helical backbone.

Helical wheel projection

View down an α-helix axis showing side-chain positions; each residue rotates about 100°.

Amphipathic α-helix

Helix with hydrophobic residues clustered on one face and polar or charged residues on the other.

Why can glycine destabilize α-helices?

Its unusually high backbone flexibility makes a regular helix less favorable.

Why can proline disrupt α-helices?

Its ring restricts φ and its backbone N lacks an H-bond donor, often causing a kink.

β-sheet

Extended β-strands held together by hydrogen bonds between backbone C=O and N–H groups.

β-strand φ and ψ angles

Approximately φ = −120° to −140° and ψ = +110° to +140°.

Parallel β-sheet

Adjacent β-strands run in the same N→C direction; hydrogen bonds are angled or offset.

Antiparallel β-sheet

Adjacent β-strands run in opposite N→C directions; hydrogen bonds are more nearly linear.

β-sheet side-chain orientation

Side chains alternate above and below the sheet; residues i and i+2 face the same side.

β-strand rise per residue

Approximately 3.5 Å along the strand.

Why are β-sheets pleated and twisted?

Allowed backbone geometry produces a pleated strand; real sheets usually twist slightly.

β-hairpin

Two antiparallel β-strands connected by a short reversal, often a β-turn.

β–α–β connection

Longer crossover connecting parallel β-strands through an intervening α-helix.

β-turn

Four-residue segment (i to i+3) that reverses the direction of the polypeptide chain.

β-turn hydrogen bond

Often forms between the C=O of residue i and N–H of residue i+3.

Type I β-turn

Common β-turn geometry in which proline often occupies position i+1.

Type II β-turn

Proline often occurs at i+1; glycine favored at i+2 due to required positive φ.

Polyproline II (PPII)

Extended left-handed helix with about 3 residues per turn and no regular intrachain backbone H-bonds.

PPII φ and ψ angles

Approximately φ = −75° and ψ = +145°.

Collagen Gly–X–Y repeat

Every third residue is Gly; X is often Pro and Y is often 4-hydroxyproline.

Why must collagen contain glycine every third residue?

Glycine's tiny side chain allows tight packing in the triple-helix center.

Collagen triple helix

Three left-handed PPII-like chains assemble into one right-handed triple helix.

Collagen triple-helix stabilization

Interchain backbone hydrogen bonds, tight packing and Pro/Hyp conformational effects.

Collagen post-translational hydroxylation

Selected Pro and Lys residues are hydroxylated after protein synthesis.

4-Hydroxyproline (Hyp)

Modified proline that helps stabilize the collagen triple helix.

5-Hydroxylysine (Hyl)

Modified lysine that may be glycosylated and contributes to later collagen cross-linking.

Collagen hydroxylase requirements

Fe²⁺, O₂, α-ketoglutarate and vitamin C.

Vitamin C deficiency and collagen

Inadequate Pro/Lys hydroxylation weakens collagen and causes scurvy.

Collagen assembly sequence

Individual α chains → modifications → triple helices → fibrils → covalent cross-links.

Secondary-structure propensity

Statistical tendency of residues to favor helices, strands or turns; depends on sequence context.

Protein tertiary structure

Overall 3D arrangement of one polypeptide, including helices, sheets, loops and side chains.

Forces stabilizing tertiary structure

Hydrophobic effect, van der Waals contacts, hydrogen bonds, electrostatics and sometimes disulfides.

Protein native-state ensemble

Dynamic group of closely related conformations near a favorable free-energy minimum.

Protein domain

Compact structural region that can often fold relatively independently and perform a distinct function.

Protein motif vs. domain

Motif is a recurring arrangement of secondary structures; a domain is a larger, often independently folding unit.

X-ray crystallography

Produces an electron-density map into which an atomic model is fitted.

Crystallographic resolution

Reported in Å; smaller values mean greater structural detail.

Solution NMR

Uses nuclear signals and structural restraints to generate an ensemble of solution conformations.

Cryo-electron microscopy (cryo-EM)

Rapidly vitrifies samples and reconstructs 3D maps from many 2D particle images.

β–α–β motif

Two parallel β-strands connected through an α-helix.

β-hairpin motif

Two antiparallel β-strands joined by a short turn.

Greek-key motif

Four β-strands connected in a characteristic folded topology.

α–α motif

Recurring arrangement of two packed α-helices.

β-barrel

Curved β-sheet closed into a cylindrical structure by hydrogen bonding between strands.

Soluble vs. membrane β-barrels

Soluble barrels often face hydrophobic residues inward; membrane barrels often face them outward toward lipids.

Rossmann fold

Nucleotide-binding architecture with a parallel β-sheet flanked by α-helices, built partly from β–α–β motifs.

GAPDH domains

N-terminal NAD⁺-binding Rossmann domain and C-terminal catalytic domain.

Globular protein side-chain organization

Hydrophobic residues usually buried; polar and charged residues often exposed to water.

Why can buried polar residues be stable?

They form internal H-bonds, salt bridges or functional interactions compensating for lost solvation.

Cytochrome c heme coordination

His18 and Met80 coordinate heme iron; Cys14 and Cys17 attach to heme via thioether bonds.

Hydropathy

Relative preference of a residue for nonpolar versus aqueous environments.

Positive vs. negative hydropathy

Positive values indicate more hydrophobic character; negative values indicate more hydrophilic character.

Hydropathy plot

Moving-window average across a sequence; long hydrophobic stretches can suggest membrane-spanning segments.

Salt bridge

Electrostatic interaction between oppositely charged side chains that can stabilize a protein fold.

C2H2 zinc finger

Compact ββ–α motif stabilized by tetrahedral Zn²⁺ coordination with two Cys and two His residues.

Zinc finger function

Often recognizes DNA; some zinc-finger families bind RNA or proteins.

Collagen lysyl oxidase

Cu²⁺-dependent enzyme that oxidizes Lys/Hyl to reactive aldehydes for extracellular cross-linking.

Why cross-link collagen?

Intermolecular covalent bonds increase fibril tensile strength and mechanical stability.

Protein structure vs. sequence conservation

Related proteins may retain similar functional 3D folds despite substantial sequence differences.

Quaternary structure

Association and interactions of two or more polypeptide subunits.

Homooligomer vs. heterooligomer

Homooligomers contain identical subunits; heterooligomers contain different subunits.

Hemoglobin quaternary structure

α₂β₂ tetramer; subunit movements contribute to cooperative oxygen binding.

Cyclic vs. dihedral protein symmetry

Cyclic: one rotational axis; dihedral: main axis plus additional twofold axes.

ATCase subunit organization

Six catalytic chains in two trimers and six regulatory chains in three dimers.

Viral capsid symmetry example

Poliovirus capsid has icosahedral symmetry built from repeating proteins.

α-keratin coiled coil

Two α-helical keratin chains associate into a coiled-coil dimer.

Keratin heptad repeat

Seven-position a–g pattern; hydrophobic residues at a and d form the coiled-coil interface.

Keratin filament assembly

Coiled-coil dimers → antiparallel staggered tetramers → ~10 nm intermediate filaments.

Why are keratin filaments nonpolar?

Antiparallel assembly of dimers cancels overall structural directionality.

ATP synthase Fo region

Membrane-associated proton-driven region with a rotating c-ring.

ATP synthase F1 region

Catalytic α₃β₃ head with ATP synthesis at β subunits around a rotating stalk.

Intrinsically disordered region

Protein segment lacking one stable tertiary structure; exists as a dynamic conformational ensemble.

Coupled folding and binding

A disordered protein region becomes more ordered when binding a partner.

Protein denaturation

Disruption of higher-order protein structure, generally without breaking peptide bonds.

Chaotropic agents

Denaturants such as urea and guanidinium chloride that favor unfolded protein conformations.

Anfinsen RNase A experiment

After removing urea and reducing agent, RNase A refolded and recovered activity.

Anfinsen's conclusion

Amino-acid sequence contains the information needed for the native fold under suitable conditions.

Scrambled disulfides

Incorrect disulfide pairings formed before proper folding, leaving RNase A inactive.

Co-translational folding

Protein folding can begin as a growing polypeptide emerges from the ribosome.