4.1 Principles of protein folding and stability

Proteins are complex molecules with intricate structures that determine their functions. From the primary sequence of amino acids to the final 3D shape, proteins fold through a series of hierarchical levels. This folding process is driven by various forces and interactions.

Understanding protein folding is crucial for grasping how proteins work in living systems. We'll explore the driving forces behind folding, the role of chaperones in assisting the process, and challenges like denaturation. We'll also discuss key concepts like Anfinsen's dogma and the Levinthal paradox.

Protein Structure Hierarchy

Primary Structure

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• Sequence of amino acids linked together by peptide bonds
• Determined by the genetic code in DNA and RNA
• Includes any disulfide bonds between cysteine residues
• Provides the foundation for higher levels of protein structure (secondary, tertiary, quaternary)

Secondary Structure

• Local folding patterns of the polypeptide chain
• Stabilized by hydrogen bonds between amino acid residues
• Common secondary structures include alpha helices and beta sheets
  • Alpha helix: coiled structure with hydrogen bonds between every fourth amino acid (3.6 residues per turn)
  • Beta sheet: extended structure with hydrogen bonds between adjacent polypeptide strands (parallel or antiparallel)
• Secondary structure is determined by the primary sequence of amino acids

Tertiary and Quaternary Structure

• Tertiary structure: three-dimensional folding of a single polypeptide chain
  • Stabilized by interactions between side chains of amino acids (hydrophobic interactions, hydrogen bonds, ionic bonds, disulfide bonds)
  • Gives proteins their unique shapes and functions (active sites, binding pockets)
• Quaternary structure: association of multiple folded polypeptide subunits
  • Stabilized by the same interactions as tertiary structure between subunits
  • Examples include hemoglobin (four subunits) and DNA polymerase (multiple subunits)

Protein Folding Driving Forces

Hydrophobic Effect

• Tendency of nonpolar amino acid side chains to cluster together in the interior of a protein
• Driven by the unfavorable interaction between nonpolar groups and water
• Minimizes the surface area of hydrophobic residues exposed to water
• Major driving force for protein folding and stability

Hydrogen Bonding and Disulfide Bonds

• Hydrogen bonds: electrostatic attraction between a hydrogen atom bonded to an electronegative atom (N, O) and another electronegative atom
  • Stabilizes secondary structures (alpha helices, beta sheets)
  • Also contributes to tertiary and quaternary structure
• Disulfide bonds: covalent bonds between the sulfur atoms of two cysteine residues
  • Provides additional stability to protein structure
  • Can link different parts of a polypeptide chain or different subunits

Protein Folding Assistance and Challenges

Chaperones

• Proteins that assist in the folding of other proteins
• Prevent aggregation of unfolded or misfolded proteins
• Examples include heat shock proteins (Hsp60, Hsp70) and chaperonins (GroEL/GroES)
• Chaperones do not contain information for the final folded state of a protein

Protein Denaturation

• Loss of native protein structure due to disruption of stabilizing interactions
• Can be caused by changes in temperature, pH, or chemical denaturants (urea, guanidinium chloride)
• Often results in loss of protein function
• Denaturation can be reversible or irreversible depending on the severity of the denaturing conditions

Anfinsen's Dogma and Levinthal Paradox

• Anfinsen's dogma: the amino acid sequence of a protein determines its final folded state
  • Demonstrated by refolding of denatured ribonuclease A in vitro
  • Implies that protein folding is a spontaneous process driven by the amino acid sequence
• Levinthal paradox: the vast number of possible conformations a protein can adopt makes it impossible to find the native state by random search
  • Suggests that protein folding must follow specific pathways or folding funnels to reach the native state quickly
  • Highlights the role of chaperones and intermediate folding states in guiding the folding process