Overview
This lecture covers protein denaturation, methods of denaturing proteins, factors affecting protein stability, and the concept of melting temperature (Tm) as it relates to protein folding and stability.
Protein Denaturation and Stability
- Protein denaturation is the unfolding of proteins, often used to assess protein stability.
- Melting temperature (Tm) is the temperature at which a protein is 50% denatured (half unfolded).
- Higher Tm indicates greater protein stability.
- Denaturation involves a transition from the native folded state to the unfolded state.
- The average energy required to denature a protein is about 0.4 kJ/mol per amino acid.
Methods of Protein Denaturation
- Heat: Raising temperature unfolds proteins by disrupting interactions.
- pH Changes: Altering pH can disrupt charge interactions, protein-dependent in effectiveness.
- Detergents (e.g., SDS): Amphipathic molecules disrupt hydrophobic interactions and unfold proteins.
- Chaotropic Agents: Small molecules like urea or guanidinium ion disrupt hydrophobic effect and hydrogen bonding, causing unfolding.
Effects and Reversibility of Denaturation
- Most denaturation methods are reversible; removing the denaturing condition often allows the protein to refold.
- Proteins may not always refold perfectly after denaturation, but they often return to their native structure.
Measuring Denaturation
- Protein denaturation can be measured by plotting a melting curve (percent unfolded vs. temperature or chaotropic agent concentration).
- Tm can also be determined by concentration of chaotropic agents (less common than temperature).
Key Terms & Definitions
- Denaturation — Loss of a protein’s native three-dimensional structure.
- Melting Temperature (Tm) — Temperature at which a protein is 50% unfolded.
- Apo Protein — Protein lacking its prosthetic (non-protein) group.
- Detergents — Amphipathic molecules (e.g., SDS) that disrupt protein structure by interacting with hydrophobic regions.
- Chaotropic Agents — Small molecules (e.g., urea, guanidinium ion) that disrupt hydrophobic and hydrogen bonding interactions, promoting unfolding.
Action Items / Next Steps
- Review the role and behavior of chaotropic agents, especially urea and guanidinium ion.
- Prepare to learn about protein folding pathways in the next lecture.