Biochem CH.4 Protein Structures

What is the chemical nature of the peptide bond and how does resonance influence its properties?

The peptide bond is a resonance hybrid of two canonical structures (C=O–N ↔ C–O⁻=N⁺). Resonance gives it partial double-bond character, making it rigid and planar, less reactive than an ester, and contributing a large dipole moment in the favored trans configuration.

Why is rotation around the peptide bond restricted?

Because of the partial double-bond character from resonance, the C–N peptide bond cannot freely rotate, forcing the backbone into planar peptide units linked at α-carbons.

Which bonds of the backbone allow rotation, and how are these rotations named?

Rotation is possible only around the N–Cα bond (φ, phi angle) and the Cα–C (carbonyl) bond (ψ, psi angle). Rotation around the peptide C–N bond itself is restricted.

What are the φ and ψ angle values in a fully extended polypeptide?

Both φ and ψ are approximately +180° in a fully extended polypeptide.

How does steric hindrance affect allowed φ and ψ combinations?

Many φ/ψ combinations are sterically disallowed because of clashes between backbone or side-chain atoms. Only certain regions permit stable H-bonding and packing, giving rise to common secondary structures.

What is a Ramachandran plot and what does it show?

It is a graph of allowed φ (x-axis) vs. ψ (y-axis) angles for amino acid residues in proteins. It shows:

• Common secondary structures (α-helices, β-sheets, etc.)

• Forbidden regions due to steric hindrance

• Regions of unusual but allowed conformations.

Which regions of a Ramachandran plot correspond to common secondary structures?

• Right-handed α-helix: φ ≈ –60°, ψ ≈ –60°

• Left-handed α-helix: φ ≈ +60°, ψ ≈ +60°

• β-sheets (antiparallel/parallel): φ ≈ –120°, ψ ≈ +120°

• Right-twisted β-sheets: φ ≈ 100–120°, ψ slightly >120°

• Collagen triple helix: φ ≈ –60°, ψ ≈ +150°

Describe the geometry and bonding of the α-helix.

A right-handed helix stabilized by hydrogen bonds between the carbonyl oxygen of residue i and the amide hydrogen of residue i+4. It has 3.6 residues per turn, a pitch of 5.4 Å, peptide bonds aligned parallel to the helix axis, and side chains projecting outward roughly perpendicular to the axis.

What is the inner and outer diameter of an α-helix and why is this important?

• Inner diameter (without side chains): 4–5 Å → too small for even a water molecule.

• Outer diameter (including side chains): 10–12 Å → fits neatly into the major groove of B-form DNA, enabling DNA–protein recognition.

Why do residues i and i+7 (or i and i+8) align on top of each other?

Because the helix has 3.6 residues per turn, a repeat of about 7 residues spans ~2 full turns, bringing i and i+7/i+8 nearly into vertical alignment.

Which amino acids are strong α-helix formers and why?

Alanine (best), Leucine, Methionine, Lysine, Arginine – small or unbranched side chains minimize steric clash and favor ideal φ/ψ angles. Ala in particular has a very low ΔΔG for helix formation.

Which amino acids are helix breakers and why?

• Proline: ring locks φ, preventing rotation and lacks the N–H needed for i→i+4 H-bonding.

• Glycine: very small and flexible, providing too much conformational freedom and favoring coils or turns instead.

How can side-chain charge and bulk disrupt an α-helix?

• Like charges (e.g., many Glu⁻ or many Lys⁺ at pH 7) repel, destabilizing the helix.

• Bulky or branched residues (Asn, Ser, Thr, Cys) in close proximity cause steric clash.

• Aromatic residues spaced three residues apart can stabilize through hydrophobic stacking.

What is the ΔΔG° scale for α-helix formation?

A small ΔΔG° (near 0, e.g., Ala) means strong helix formation; large ΔΔG° (e.g., Gly ~4.6, Pro >4) means poor helix formation because the free-energy cost of adopting α-helical geometry is high.

What is the helix macrodipole and why is it significant?

Each peptide bond is polar (C=O negative, N–H positive). In an α-helix these dipoles align to produce a large overall dipole with a partial positive charge at the N-terminus and partial negative charge at the C-terminus.
Negatively charged residues (Asp⁻, Glu⁻) are often found near the N-terminal end and positively charged residues (Lys⁺, Arg⁺) near the C-terminal end to stabilize this dipole.

What sequence pattern creates an α-helix with one hydrophobic face and one hydrophilic face?

A sequence with hydrophobic residues every 3–4 positions so they line up on one side, e.g.
Leu–Ala–Lys–Leu–Ala–Lys–Leu,
producing an amphipathic helix useful in membrane or protein–DNA interactions.

What structural features give a β-sheet its pleated appearance?

The planarity of the peptide bond and the tetrahedral geometry of the α-carbon force the backbone into a zigzag arrangement. When multiple strands hydrogen-bond side by side, the sheet is “pleated,” with side chains alternating up and down.

How are β-sheets stabilized?

By inter-strand hydrogen bonds between backbone carbonyl oxygens and amide hydrogens of adjacent strands.

What is the difference between parallel and antiparallel β-sheets?

• Parallel β-sheets: strands run in the same N→C direction; hydrogen bonds are bent → weaker.

• Antiparallel β-sheets: strands run in opposite directions; hydrogen bonds are straight/in-line → stronger and more stable.

How do side chains project in a β-sheet?

They protrude alternately above and below the sheet plane, helping stabilize packing and interactions.

What is the function of a β-turn?

To reverse the direction of a polypeptide chain (about 180°) over only four residues, allowing compact, globular folding and connecting adjacent β-strands.

What stabilizes a β-turn?

A hydrogen bond between the carbonyl oxygen of residue 1 and the amide hydrogen of residue 4 (i → i+3 bond).

Which amino acids are common in β-turns and why?

• Proline (position 2): ring rigidity locks the φ angle and enforces the bend.

• Glycine (position 3): tiny side chain provides flexibility to accommodate the tight dihedral angles.

• Polar residues (Asn, Ser, Asp) often help by hydrogen-bonding with water on the protein surface.

Describe Type I vs. Type II β-turns.

• Type I: more common; proline often at position 2; residue 3 can be any amino acid; more compact.

• Type II: less common; glycine almost always at position 3 to relieve steric strain; position 2 can vary.

What are “physical links” in β-turns?

The peptide backbone of a β-turn is continuous with its β-strand, so a single chain can flow from one strand, through the turn, and into the next, providing a flexible hinge and stabilizing sheet architecture.

What is unusual about peptide bonds involving proline?

~6 % of peptide bonds preceding proline adopt the cis configuration (vs. >99.9 % trans for others). This cis form is often found in β-turns.

How is proline isomerization catalyzed?

By prolyl isomerases, which accelerate interconversion between cis and trans proline peptide bonds to assist folding.

Which amino acids strongly favor α-helices?

Glu, Met, Ala, Leu, Lys – small, unbranched, or charged groups that fit the helical geometry and make favorable H-bonds.

Which amino acids strongly favor β-sheets?

Val, Ile, Tyr, Phe, Trp, Thr – bulky, branched, or aromatic side chains that pack well in extended sheet geometry and form stabilizing side-to-side interactions.

Which amino acids favor β-turns?

Gly, Asn, Ser, Asp, Pro – small, flexible, or rigid side chains suited for tight turns and surface exposure.

What does circular dichroism (CD) measure?

It measures the difference in absorption (Δε) of left- and right-circularly polarized light by peptide chromophores, which depends on the protein’s secondary structure.

How do CD spectra distinguish α-helix, β-sheet, and random coil?

• α-helix: Strong negative peaks at 208 nm and 222 nm, strong positive near 190 nm.

• β-sheet: Negative near 215 nm, positive near 195 nm.

• Random coil: Weak overall signal, slight negative near 195 nm.

How can CD be used to estimate a protein’s folding or unfolding?

By comparing a protein’s CD spectrum to reference spectra (α-helix vs. β-sheet vs. random coil), scientists can estimate secondary-structure content and monitor folding or denaturation in real time.

Define protein tertiary structure and what stabilizes it.

Tertiary structure is the overall 3-D arrangement of all atoms in a single polypeptide.
Stabilized mainly by numerous weak interactions between side chains—hydrophobic interactions, hydrogen bonds, ionic (electrostatic) interactions, and sometimes covalent disulfide bonds.
Residues that interact in tertiary structure need not be adjacent in the primary sequence.

What are the two major classes of tertiary structure?

• Fibrous proteins: long strands or sheets; usually dominated by one type of secondary structure; structural/support roles.

• Globular proteins: compact, roughly spherical; contain multiple secondary-structure types; enzymes, transport, regulation.

How do fibrous proteins differ structurally and functionally from globular proteins?

Fibrous proteins are elongated, insoluble due to high hydrophobic content and simple repeating secondary structures. They mainly provide support, strength, shape, and external protection (e.g., keratin in hair, collagen in tendons).
Globular proteins are soluble, compact, and chemically active, suited to catalysis and transport.

Name the three major secondary-structure motifs in fibrous proteins and give an example of each

Name the three major secondary-structure motifs in fibrous proteins and give an example of each

Describe the basic α-keratin structural hierarchy.

• Single polypeptide: right-handed α-helix.

• Two-chain coiled coil: two α-helices wrap around each other in a left-handed supercoil.

• Protofilament: two coiled coils pack side-by-side (20–30 Å diameter).

• Protofibril: four protofilaments assemble; many protofibrils bundle into the visible keratin fiber

What interactions stabilize α-keratin structure?

• Hydrophobic striping: hydrophobic residues appear every 3–4 residues and align along one face to drive coiled-coil formation.

• Disulfide bonds (cysteine–cysteine): covalent cross-links that add strength and determine hardness (more S–S = harder structures like nails or horns).

Why is α-keratin insoluble?

Its surface is rich in hydrophobic residues, which are buried through tight packing of many polypeptide chains into supramolecular fibers, preventing water penetration.

What secondary structure dominates silk fibroin, and how is it stabilized?

Silk fibroin consists of antiparallel β-sheets.
Stabilization comes from:

• Inter-sheet hydrogen bonding between backbone amide and carbonyl groups.

• London dispersion forces between closely packed small side chains (Ala, Gly), allowing tight interdigitation.

What mechanical properties result from silk fibroin’s structure?

It is strong yet flexible—the β-sheets give tensile strength, and the weak inter-sheet interactions permit flexibility.

How does spider silk achieve both high strength and elasticity?

Spider silk is a composite:

• Crystalline β-sheet regions (fibroin-rich) provide steel-like strength.

• Rubber-like amorphous regions allow significant stretch before breaking.

Where is collagen found and what is its role?

Collagen is the major structural protein of connective tissue—tendons, ligaments, cartilage, bone matrix, cornea—and provides high tensile strength without stretch.

Describe the primary and secondary structure of a single collagen α-chain.

Each α-chain is a left-handed helix (not an α-helix) with 3 amino acids per turn.
Its repeating tripeptide motif is Gly–X–Pro or Gly–X–4-hydroxyproline (Hyp).
Glycine’s minimal side chain (H) is essential every third residue to allow tight packing.

Explain how three collagen chains assemble into the triple helix.

Three left-handed α-chains wind around each other to form a right-handed triple helix ~300 nm long and 1.5 nm thick.
This is the fundamental subunit of collagen fibrils.

How are collagen fibrils formed and strengthened?

Triple-helical collagen molecules align in a staggered, parallel fashion and are covalently cross-linked (via lysine or hydroxylysine side chains).
The staggered alignment produces 64 nm cross-striations visible by EM and gives very high tensile strength.

What is 4-hydroxyproline and why is it critical for collagen?

4-hydroxyproline is a post-translationally hydroxylated form of proline.
It adds extra hydrogen bonding between chains and forces proline into the proper “exo” conformation, stabilizing the triple helix.

Which enzyme and cofactors are required to produce 4-hydroxyproline, and what disease results from their deficiency?

The enzyme prolyl-4-hydroxylase requires α-ketoglutarate, molecular O₂, and ascorbate (vitamin C).
Vitamin C deficiency impairs hydroxylation, weakening collagen and causing scurvy (bleeding gums, poor wound healing).

Compare the mechanical strength of a collagen triple helix to steel.

A collagen triple helix has greater tensile strength than a steel wire of equal cross-section, allowing connective tissues to resist stretching forces.

What is a globular protein and how does its structure differ from a fibrous protein?

Globular proteins fold into compact, roughly spherical shapes and typically contain multiple secondary structures (α-helices, β-sheets, turns). They are water-soluble, with hydrophobic residues buried inside and hydrophilic residues on the surface. Functions include enzymes, transport, regulation, and signaling, unlike fibrous proteins, which are elongated, insoluble, and primarily structural.

Name two examples of globular proteins and their functions.

• Myoglobin (PDB ID 1MBO): oxygen storage and transport in muscle.

• Human serum albumin (Mr ~64 kDa): transports hormones, fatty acids, and maintains blood osmotic pressure.

How is the interior and exterior of a typical globular protein organized?

• Interior: tightly packed, predominantly hydrophobic side chains, stabilized by van der Waals forces.

• Exterior: mostly hydrophilic residues forming hydrogen bonds with water, stabilizing solubility.

Define a protein motif (also called a fold).

A recurring combination of secondary structures (e.g., α-helices, β-sheets) arranged in a specific 3D pattern that can appear in many different proteins and is associated with particular functions.

Give examples of common protein motifs.

• α-α corner

• β-α-β loop

• β barrel
  These can be building blocks of larger protein domains.

What is quaternary structure in proteins?

The association of two or more polypeptide chains (subunits) into a functional protein complex (oligomer). Subunits may be identical or different, and the overall arrangement can be symmetric or asymmetric.

Give an example of a protein with quaternary structure and describe it.

Hemoglobin is a tetramer of two α and two β subunits (or two αβ protomers). This arrangement enables cooperative oxygen binding.

Name three experimental methods to determine 3D protein structure and one key pro/con for each.

• X-ray crystallography: no size limit; requires crystallization; hydrogen positions often not visible.

• Biomolecular NMR: works in solution and detects hydrogens; best for small proteins.

• Cryo-electron microscopy: suited for very large complexes (>300 kDa); requires high purity and specialized instrumentation.

Define protein denaturation and list major causes.

Denaturation is the loss of 3D structure with loss of function. Causes include heat/cold, pH extremes, organic solvents, and chaotropic agents like urea or guanidinium chloride that disrupt hydrophobic interactions and hydrogen bonds.

Describe the classic ribonuclease refolding experiment and its significance

Christian Anfinsen denatured ribonuclease with urea + 2-mercaptoethanol (breaking disulfide bonds). Upon removal of these agents, the protein spontaneously refolded and regained activity, proving that primary sequence alone determines native 3D structure. This earned him the 1972 Nobel Prize.

Outline the general pathway of protein folding.

Folding is hierarchical:

1. Rapid formation of local secondary structures (α-helices, β-sheets).

2. Assembly of supersecondary structures (motifs/domains).

3. Final tertiary structure with minimal free energy.
   Folding is cooperative and decreases conformational entropy.

Differentiate molecular chaperones from chaperonins

• Chaperones (e.g., Hsp70, Hsp90): bind exposed hydrophobic patches to prevent aggregation and assist folding in the open cytosol; do not form a sealed folding chamber.

• Chaperonins (e.g., GroEL/GroES in bacteria, Hsp60/Hsp10 in mitochondria): form a double-ring barrel with a cap, providing an isolated folding compartment where ATP-driven cycles promote proper folding.

How does the Hsp60/Hsp10 (GroEL/GroES) system fold proteins?

• Unfolded protein enters the Hsp60 barrel.

• ATP binding triggers Hsp10 capping, creating a sealed cavity.

• Protein folds inside the isolated chamber.

• ATP hydrolysis releases the cap and the properly folded protein.

Where do proteins fold if only Hsp70 (and not a chaperonin) assists?

Folding occurs directly in the cytosol or organelle lumen, with Hsp70 repeatedly binding and releasing hydrophobic segments to prevent aggregation and allow the protein to fold naturally in the aqueous environment.

How can protein misfolding lead to disease?

Misfolded proteins can expose hydrophobic or β-sheet-rich regions, leading to aggregation into amyloid fibrils. These insoluble fibrils accumulate as plaques in tissues, damaging cells and impairing organ function.

Describe the formation of amyloid fibrils.

• Partially unfolded proteins expose β-sheet segments.

• These segments stack via hydrogen bonds into protofilaments with cross-β structure.

• Protofilaments laterally associate into amyloid fibrils, which are highly stable and resistant to degradation.

Give an example of a disease caused by amyloid fibril formation.

Alzheimer disease, where misfolded amyloid-β peptides aggregate into fibrils and form extracellular plaques in the brain, disrupting neuronal communication.

Besides Alzheimer’s, name other disorders associated with protein misfolding and amyloid formation.

Examples include Parkinson’s disease (α-synuclein aggregates), Huntington’s disease (polyglutamine expansions), and prion diseases (misfolded PrP protein).