Membrane lecture 2

Question 1

Expression

Answer 1

• Need ↑ amounts
• Over-expression can be tricky
• Multiple sequence alignment to look for bacterial homologue

Question 2

Detergents

Answer 2

• Keeps membrane protein in soluble form
• Needs to be sufficiently disruptive to remove phospholipid but x Δ conf of protein
• Most have hydrophilic head (makes water soluble) + non-polar tail, bile acid has both polar + non-polar ‘faces’
• octyl glucoside = useful, DDM = good
• Micellisation

Question 3

Crystallisation

Answer 3

• Lipid cubic phase (curved 3D liquid crystalline structure that self-assembles, stabilises proteins)
• Monoolein = often used

Question 4

Alternatives to crystallisation

Answer 4

• Amphipols = polymers w/ hydrophobic + hydrophilic regions
• Nanodisc = used in cryo-EM , scaffold protein forms 2 belts that make stable environment, incorporate protein inside
• nanobody = add H20-soluble protein

Question 5

X ray diffraction

Answer 5

• Hard to crystallise
• Detergents means have weak lactic forces so ↓ ordered, ↓ resolution
• Nanobody/lipid cubic phase

Question 6

Cryo-EM

Answer 6

• Single particle EM, freeze + look at structure, 3D info
• Statistical sorting
• 3.3A resolution
• Shorter time

Question 7

Solution NMR

Answer 7

• Small membrane proteins that x crystallise + too small for cryo-EM
• Solubilise w/ detergent, different structure

Question 8

MD

Answer 8

• Simulate flexibility of protein at room temperature

- Can look at interactions in cell-like environment