The Three-Dimensional Structure of Proteins

Linus Pauling’s Principles for Secondary Structures

Linus Pauling established foundational principles that govern the formation of protein secondary structures, ensuring their stability and regularity.

• Planarity of the Peptide Bond: The peptide bond is planar due to partial double-bond character, restricting rotation and stabilizing the backbone.

• Allowed Rotations: Only the phi (φ) and psi (ψ) angles of the polypeptide backbone can rotate, dictating possible conformations.

• Hydrogen Bonding: Regular patterns of hydrogen bonding between backbone amide and carbonyl groups stabilize secondary structures.

• Steric Constraints: Atoms in the backbone and side chains must avoid steric clashes, limiting possible conformations.

Levels of Protein Structure

Proteins exhibit hierarchical structural organization, each level stabilized by specific interactions.

• Primary Structure (1°): The linear sequence of amino acids in a polypeptide chain, linked by peptide bonds.

• Secondary Structure (2°): Local folding patterns stabilized by hydrogen bonds, such as α-helices and β-sheets.

• Tertiary Structure (3°): The overall three-dimensional shape of a single polypeptide, stabilized by hydrophobic interactions, hydrogen bonds, ionic interactions, and disulfide bonds.

• Quaternary Structure (4°): The arrangement of multiple polypeptide subunits in a multi-subunit protein, stabilized by non-covalent interactions and sometimes disulfide bonds.

Types of Interactions at Each Level

• Primary: Covalent peptide bonds.

• Secondary: Hydrogen bonds between backbone atoms.

• Tertiary: Hydrophobic interactions, hydrogen bonds, ionic bonds, van der Waals forces, disulfide bridges.

• Quaternary: Non-covalent interactions (hydrophobic, ionic, hydrogen bonds), sometimes covalent (disulfide bonds).

Peptide Bond: Definition and Properties

The peptide bond is a covalent bond formed between the carboxyl group of one amino acid and the amino group of another, releasing water (a condensation reaction).

• Planarity: The peptide bond is planar due to resonance, restricting rotation.

• Partial Double-Bond Character: The C-N bond has partial double-bond character, making it rigid.

• Trans Configuration: Most peptide bonds are in the trans configuration, minimizing steric hindrance.

Identifying Peptide Bonds: In a polypeptide, peptide bonds link the carbonyl carbon of one residue to the amide nitrogen of the next.

Parts of a Polypeptide

• N-terminus: The end of the polypeptide with a free amino group.

• C-terminus: The end with a free carboxyl group.

• Amino Acid Residue: An amino acid unit within a polypeptide chain, after condensation.

Psi (ψ) and Phi (φ) Bonds

• Phi (φ) Angle: Rotation around the N–Cα bond.

• Psi (ψ) Angle: Rotation around the Cα–C bond.

• These angles determine the backbone conformation and are restricted by steric hindrance.

Stereochemistry of the Peptide Bond

• Peptide bonds are almost always in the trans configuration, except for some involving proline.

• The planarity and configuration affect protein folding and stability.

Secondary Structure

Secondary structures are regular, repeating local structures stabilized by hydrogen bonds.

α-Helix

• Right-handed coil with 3.6 residues per turn.

• Hydrogen bonds form between the carbonyl oxygen of residue i and the amide hydrogen of residue i+4.

• Side chains project outward from the helix axis.

Factors Destabilizing α-Helix:

• Proline residues (introduce kinks)

• Glycine residues (too flexible)

• Electrostatic repulsion between adjacent charged side chains

• Bulkiness of adjacent side chains

β-Sheet

• Composed of β-strands connected laterally by hydrogen bonds.

• Can be parallel (strands run in the same direction) or antiparallel (strands run in opposite directions).

• Side chains alternate above and below the plane of the sheet.

Parallel vs. Antiparallel β-Sheets

Fibrous Proteins

Fibrous proteins are elongated, insoluble proteins with structural roles.

• Keratin: Predominantly α-helix; found in hair, nails.

• Fibroin: Predominantly β-sheet; found in silk.

• Collagen: Triple helix structure; unique to collagen fibers in connective tissue.

Super Secondary Structure, Motifs, and Domains

• Super Secondary Structure: Combinations of secondary structures forming recognizable patterns (e.g., β-α-β motif).

• Motif: A specific arrangement of secondary structures with a particular function or structural role (e.g., helix-turn-helix).

• Domain: A distinct, independently folding unit within a protein, often associated with a specific function.

• β-Turn (Hairpin Turn): A short turn that reverses the direction of the polypeptide chain, often connecting strands of antiparallel β-sheets.

Globular Proteins

Globular proteins are compact, generally soluble proteins with diverse functions.

• Hydrophobic amino acids are typically buried in the protein core.

• Hydrophilic amino acids are exposed on the surface, interacting with the aqueous environment.

Anfinsen Experiment and Protein Folding

The Anfinsen experiment with ribonuclease A demonstrated that the information required for protein folding is contained in the primary sequence.

• Ribonuclease A was denatured and then allowed to refold, regaining its activity.

• This showed that protein folding is spontaneous and determined by the amino acid sequence.

Thermodynamics of Protein Folding

Protein folding is governed by thermodynamic principles, seeking the lowest free energy state.

• ΔG (Gibbs Free Energy): Folding is favorable when ΔG is negative.

• Hydrophobic Effect: Nonpolar residues cluster in the core, increasing entropy of water and stabilizing the folded state.

• Disulfide Bonds: Covalent bonds between cysteine residues stabilize the folded structure.

Molten Globule Theory of Protein Folding

The molten globule is an intermediate state in protein folding, characterized by native-like secondary structure but a dynamic, flexible tertiary structure.

• Represents a partially folded state en route to the fully folded, functional protein.

• Allows for rapid sampling of conformations before final stabilization.

Example: During folding, a protein may first collapse into a molten globule before achieving its final, stable conformation.