Lecture Notes: Ribosome Experiments and Protein Folding

Overview

• Exploration of ribosome experiments aimed at understanding protein translation, particularly involving stop codon suppression.
• Introduction to protein folding, focusing on the process post-ribosome.

Ribosome Experiment Recap

• Previous Topic: Incorporating unnatural amino acids using new ribosomes.
• Orthogonal Ribosome: Designed for binding orthogonal mRNA to address amber stop codon suppression.
• Problem: Release factor 1 causing truncated protein phenotypes.

Experiments on Ribosome Efficiency

1. Protein Yield: Comparing translation efficiency to starting orthogonal ribosome.
2. Amino Acid Misincorporation:
   • GST-MBP fusion protein used with a protease cleavage site.
   • Radiolabeled cysteine as a probe in experiments to detect misincorporation.
   • Results showed similar protein synthesis across various systems.

Experiment Details

• Proteins Analyzed:
  • GST and MBP fragments separated using SDS PAGE.
  • Coomassie stain for total protein vs. radioactivity analyses.
• Findings:
  • All systems showed similar protein yield.
  • Rybax ribosome showed efficient cysteine incorporation.
  • Limitation: Only tested cysteine, not other amino acids.

Protein Folding Module

• Introduction to Protein Folding: How polypeptides fold into native structures post-ribosome translation.

Concepts in Protein Folding

• Macromolecular Crowding: Cellular environment is crowded, influencing protein folding.
• Chaperones: Assist in the proper folding of proteins. Protect against misfolding and aggregation.

Folding in Different Environments

• Cytoplasm: Focus on how proteins fold with the help of chaperones.

Key Questions

• How do proteins achieve their native folds?
• What machinery assists folding?
• Relationship between folding and disease.

Protein Folding Experiments

• Anfinsen’s Hypothesis: Primary sequence dictates the final shape.
• Experiment with Ribonuclease A demonstrated refolding to native form post-denaturation.
• Levinthal’s Paradox: Explores the complexity of protein folding.

Energy Landscapes

• Folding represented as an energy landscape with barriers and native states.
• Chaperones help traverse these landscapes to avoid non-productive folding.

Experimental Methods

• Techniques to study folding: Fluorescence, circular dichroism, NMR, etc.
• Denaturation methods: Heat, pH, chemical agents like urea.

Chaperones and Folding Machines

• Chaperones: Aid in preventing misfolding and assist in achieving correct fold.
• Examples: GroEL, GroES, DNA KJ, Trigger Factor.

Protein Misfolding and Disease

• Misfolded proteins can lead to diseases: Alzheimer's, Parkinson's, ALS.
• Aggregates, oligomers, and fibrils contribute to disease pathology.

Conclusion

• Proper protein folding is crucial for cellular function.
• Folding involves a complex interplay of structural biology, thermodynamics, and cellular machinery.
• Future discussions will cover chaperones and their roles in vivo and in vitro.

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These notes summarize key points from a lecture on ribosome experiments and protein folding, highlighting important experiments, findings, and theoretical concepts in molecular biology.