Lecture Notes: Membrane Protein Topology

Overview

• Focus on secreted and transmembrane proteins
• Understanding membrane protein topology and insertion
• Experiments to determine protein looping and topology

Transmembrane Protein Diversity

• Proteins can have varied topologies:
  • Amino terminus inside lumen, carboxy terminus outside
  • Vice versa (flipped topologies)
  • Amino terminus as a transmembrane domain
• Multiple membrane-spanning proteins (e.g., CFTR with 12 domains)
• Complex structures like GPI linkages

Determining Membrane Topology

Classic Experiments

1. Proteolysis

   • Tests with protease to identify external domains
   • Protease chews external domains, leaving fragments
   • Use antibodies to determine protein orientation
   • Time-consuming, reagent-intensive

2. Enzyme Tagging Assay

   • Tag proteins with enzymes (e.g., alkaline phosphatase)
   • Use enzyme location to deduce topology
   • Two scenarios:
     • Enzyme inside membrane → no substrate access
     • Enzyme outside → substrate access and color change
   • Helps determine protein topology based on enzyme position

Rules for Membrane Protein Insertion

• Odd-numbered transmembrane domains
  • Amino and carboxyl termini on opposite sides
• Even-numbered transmembrane domains
  • Amino and carboxyl termini on the same side
  • Useful with enzyme assays to map protein

Charged Amino Acids and Orientation

• Positive charges found on cytoplasmic side
• Useful for predicting protein orientation

Topogenic Sequences

• Poorly understood, guide membrane protein topology
• Signal Anchor Sequences
  • Keep transmembrane domain in channel during translation
• Stop Transfer Anchor Sequences
  • Instruct domain to exit channel, allowing continuation

Conclusion

• Understanding topology is crucial for studying protein function and mutations
• Empirical rules and experimental assays provide insight into protein orientation and insertion