BCM 251 Lecture 10: Control Over Protein Folding and Denaturation

Introduction to Protein Structure

  • Protein conformations are spatial arrangements of atoms achieved without breaking covalent bonds.
  • Native conformations are associated with specific biological activities and are typically thermodynamically stable.

Protein Folding Facts

  • Native conformation is largely determined by primary structure (Anfinsen dogma).
  • Function depends on 3D structure; misfolded proteins are dysfunctional.
  • Conformations are dynamic; denaturation means loss of structure but not every random coil is denatured.
  • Characterized by thermodynamic stability and permissible dihedral angles (Ramachandran angles).

Energy Funnel and Folding Pathways

  • Folding can be seen as a free energy funnel, moving from high free energy to native conformation.
  • Folding is hierarchical; starts with local secondary structures and progresses through hydrophobic interactions to final conformation (tertiary/quaternary).

Chaperon-Assisted Folding

  • Chaperons assist with proper protein folding and can keep proteins inactive.
  • Major families include Hsp70/Hsp40 (eukaryotes) and Hsp60 (prokaryotes).
  • They catalyze non-spontaneous folding requiring ATP.

Chaperon Functions

  • Prevent incorrect aggregation (e.g., hemoglobin in thalassemia), maintain stability of folded states under stress conditions.

Proteostasis

  • Proteostasis involves regulation of folded/unfolded proteins, protein recycling, and degradation pathways.
  • Misfolding leads to diseases like Alzheimer’s and cystic fibrosis.

Denaturation of Protein Structure

  • Denaturation involves unfolding due to physical effects (heat, pH, detergents).
  • Loss of structure leads to loss of function.

Effects of Denaturing Agents

  • Heat disrupts weak bonds; thermotolerant proteins exhibit traits resisting heat.
  • Extreme pH increases electrostatic repulsion, causing denaturation.
  • Organic solvents disrupt hydrophobic interactions and hydrogen bonds.
  • b-Mercaptoethanol reduces disulfide bonds, facilitating denaturation.

Refolding

  • Denatured proteins can refold under proper conditions (removal of detergents) demonstrating the link between primary structure and natural conformation.
  • Salting-out is used for purification, followed by dialysis to restore conformation.