Lecture 21: Quartnery Structure and Protein Folding

Protein Structure Overview

  • Hierarchical Description:

    • Primary Structure: Covalent structure, sequence of amino acids (AA).

    • Secondary Structure: Created by hydrogen bonding in the backbone, involves twists and folds of the chain.

    • Tertiary Structure: Interactions between secondary structure elements and side chains result in the overall 3D structure.

    • Quaternary Structure: Interactions among different polypeptide chains, applicable only to multimeric proteins.

Quaternary Structure

  • Importance of Quaternary Structure:

    • Many proteins lack a quaternary structure.

    • Hemoglobin: A tetramer composed of two alpha (α) and two beta (β) chains, essential for its function.

    • Restriction Enzymes: Often form homodimers that bind palindromic sequences (specific sequences that read the same forwards and backwards).

Specific Proteins

  • GAPDH:

    • Monomeric form is catalytically inactive but serves other cellular functions.

    • Tetrameric form is catalytically active, consists of 4 identical chains (homo-tetramer).

  • Collagen:

    • Most abundant protein in humans, primarily found in the extracellular matrix.

    • Sequence: Gly-X-Y, where X and Y are often proline (Pro) or hydroxyproline (Hydroxy-Pro).

    • Requires vitamin C for the modification of proline.

    • Always exists as a trimer, allowing for tight twists.

Protein Folding

  • RNase:

    • Small digestive protein that hydrolyzes RNA to nucleotides, contains 124 AA and 4 disulfide bridges.

    • Identified as a model protein for studying protein folding, with significant contributions by Christian Anfinsen (Nobel Prize, 1972).

Anfinsen’s Experiments and Protein Folding

  • Denaturation using Urea:

    • 8M Urea added to RNase disrupts hydrogen bonding and weakens hydrophobic interactions.

    • Weakening these interactions prevents clumping of hydrophobic groups, leading to renaturation.

    • Disulfide bonds temporarily disrupted during denaturation.

  • Reduction of Disulfide Bonds:

    • Addition of β-mercaptoethanol reduces and breaks disulfide bonds, critical for understanding protein folding.

  • Dialysis in Protein Refolding:

    • Removal of urea and β-mercaptoethanol through dialysis allows RNase to refold into its active structure.

    • Dialysis retains larger molecules while removing small ones (like urea).

  • Insights from Anfinsen’s Experiment:

    • Protein folding is spontaneous and influenced by the sequence of the protein.

Levinthal’s Paradox

  • The rapid folding of proteins contradicts random exploration of conformations.

  • Calculation shows that a 100 residue protein would take an unfeasible amount of time to search all conformations.

  • Suggests an ordered process of folding, not random exploration.

Protein Folding Process

  • Hydrophobic Collapse and Nucleation:

    • Initial condensation of hydrophobic regions followed by formation of short stretches of secondary structure.

  • Aggregation:

    • Involves motifs and domains forming a molten globule, leading to compaction and tertiary structure formation.

Cellular Influence on Protein Folding

  • Challenges of Protein Folding:

    • Unfolded proteins are sticky due to exposed hydrophobic surfaces.

    • Low concentrations favor folding, while high concentrations favor aggregation.

Chaperones and Chaperonins**

  • Chaperones:

    • Assist in the proper folding of proteins, preventing aggregation, and require ATP for binding and release.

  • Chaperonins:

    • Complex machinery that helps in folding partially folded polypeptides. Uses ATP hydrolysis for their function.

Protein Disulfide Isomerase (PDI)**

  • Anfinsen's discovery of PDI, which catalyzes oxidation and isomerization of disulfide bonds, aiding correct protein folding.

Proline Isomerase**

  • An enzyme that catalyzes the slow conversion between cis and trans forms of proline, facilitating proper protein configuration.

Re-evaluating Anfinsen’s Conclusions**

  • Proteins need external guidance to fold correctly, as sequence alone is insufficient.

  • Multi-domain proteins require different folding mechanisms than single-domain proteins, as seen with RNase.

Consequences of Protein Misfolding**

  • Disease Association:

    • Misfolded proteins linked to diseases such as vCJD from BSE, and neurodegenerative conditions like Alzheimer's and Parkinson's, characterized by abnormal β-sheet structures leading to aggregates.

  • Sickle Cell Anemia:

    • Hemoglobin aggregates distort cell shape, affecting a significant number of people globally, highlighting the implications of misfolded proteins in health.

Key Messages**

  • Understanding quaternary structure is essential for many protein functions.

  • Anfinsen’s experiment shaped the understanding of protein folding processes.

  • Protein folding follows an ordered process influenced by hydrophobic interactions and cellular influences, requiring a specialized chaperone machinery to prevent misfolding.