MCDB 1150 - Protein Structure Notes (Lecture 7)

Primary Structure

  • Definition: The linear sequence of amino acids in a polypeptide chain.

  • Bonds: Covalent peptide bonds linking amino acids.

    • Repeating unit: −N(H)–C(=O)–N(H)–C(=O)–-N(H)–C(=O)–N(H)–C(=O)–

  • Role: This sequence dictates all higher-level protein structures.

Secondary Structure

  • Definition: Local, repeating shapes of the polypeptide backbone.

  • Major Motifs:

    • Alpha-helix (α-helix)

    • Beta-pleated sheet (β-sheet)

    • Loop regions

  • Stabilizing Bonds: Hydrogen bonds between backbone amide and carbonyl groups. These bonds are independent of side-chain chemistry.

  • R-group Influence: Side-chain properties affect how these motifs pack into 3D structures:

    • Hydrophilic residues: Outside, water-exposed.

    • Hydrophobic residues: Inside, core.

    • Cysteine: Can form disulfide bonds (covalent, stabilizes higher structures).

Tertiary Structure

  • Definition: The complete 3D shape of a single polypeptide chain, including how its secondary structures are arranged.

  • Stabilizing Forces (R-group interactions):

    • Hydrophobic interactions: Nonpolar residues cluster in the interior.

    • Hydrogen bonds: Between side chains or side chain and backbone.

    • Ionic (electrostatic) interactions: Salt bridges between charged side chains.

    • Covalent disulfide bonds: Between cysteine residues.

  • Directionality: Defined by N-terminus and C-terminus.

  • Disulfide Bonds: Covalent links between two cysteine sulfur atoms, crucial for stabilization.

  • Example: GFP is monomeric, showing primary, secondary, and tertiary structures, but no quaternary structure.

Quaternary Structure

  • Definition: Arrangement and interactions of multiple polypeptide chains (subunits) to form a functional protein complex.

  • Terminology:

    • Homomeric: Identical subunits (e.g., homodimer).

    • Heteromeric: Different subunits.

  • Examples:

    • Cro protein: Homodimer (2 identical subunits).

    • Hemoglobin: Tetramer (2 α-globin, 2 β-globin subunits).

    • PCNA: Homotrimer (3 identical subunits forming a DNA clamp).

  • Stabilizing Forces: Same as tertiary structure (hydrophobic, hydrogen, ionic, and disulfide bonds).

  • Function: Increases functional diversity, allows cooperative effects and complex regulation.

Specific Protein Examples

  • Sickle Cell Anemia:

    • Hemoglobin (Hb): Tetramer (2α+2β2\alpha + 2\beta subunits).

    • Mutation: Hydrophilic Glutamate mutated to hydrophobic Valine in β\beta-globin.

    • Consequence: Valine creates a sticky hydrophobic patch, leading to Hb polymerization, clumping, and sickling of red blood cells under low oxygen.

  • Green Fluorescent Protein (GFP):

    • Shows primary, secondary, and tertiary structures.

    • Monomeric, thus no quaternary structure.

  • Proliferating Cell Nuclear Antigen (PCNA):

    • Homotrimer (3 identical subunits) that forms a ring around DNA, acting as a sliding clamp.

  • Arc Protein:

    • Example of protein with higher-order oligomerization (quaternary structure) involved in neuronal signaling.

Effects of Ionic Strength and Salt

  • High salt disrupts electrostatic interactions (ionic bonds, salt bridges).

  • Impact by Structure Level:

    • Primary: Largely unaffected (covalent peptide bonds).

    • Secondary: Relatively robust, but can be indirectly influenced.

    • Tertiary: Most affected, as salt bridges and ionic interactions stabilize the 3D fold.

    • Quaternary: Most susceptible; subunit interfaces often rely on electrostatic interactions, leading to dissociation.

Reversibility and Recovery After Salt Wash

  • Question: Can a salt-disrupted protein refold if salt is removed and conditions are restored?

  • Answer: Yes.

    • Primary structure remains intact (covalent peptide bonds are not broken).

    • If only noncovalent interactions were disrupted, tertiary and quaternary structures can often reform.

Summary Chart of Protein Structure Levels

  • Primary Structure:

    • Bond Type: Peptide bonds (covalent).

    • Stabilizing Interactions: Covalent peptide bonds.

    • Key Characteristic: Linear amino acid sequence; determines all higher structures.

  • Secondary Structure:

    • Bond Type: Hydrogen bonds between backbone atoms.

    • Stabilizing Interactions: Alpha-helices and beta-sheets.

    • Key Characteristic: Local folding patterns (e.g., helices, sheets).

  • Tertiary Structure:

    • Bond Type: Hydrophobic, hydrogen, ionic, and disulfide (covalent) bonds.

    • Stabilizing Interactions: 3D packing of a single polypeptide.

    • Key Characteristic: Overall 3D fold of one polypeptide chain.

  • Quaternary Structure:

    • Bond Type: Hydrophobic, hydrogen, ionic, and disulfide (covalent) bonds.

    • Stabilizing Interactions: Interactions between multiple polypeptide subunits.

    • Key Characteristic: Assembly of multiple polypeptide subunits (homo- or heteromeric).

Quick Reference Quiz Reminders

  • Secondary Structure Bonds: Hydrogen bonds between backbone atoms.

  • Salt Disruption: Tertiary and Quaternary structures (due to electrostatic interactions).

  • GFP Structure: Primary, secondary, and tertiary; not quaternary (it's monomeric).

  • Hemoglobin: Tetramer (four subunits: 2α2\alpha, 2β2\beta); shows functional complexity of quaternary structure.

  • Sickle Cell Mutation: Glu to Val substitution creates a hydrophobic patch, leading to Hb polymerization and red blood cell sickling under low oxygen.