Lecture 6 - Membranes Part 2

Question 1

what are the 3 membrane proteins?

Answer 1

• Integral
• Peripheral
• Lipid-anchored

Question 2

explain integral protein

Answer 2

• penetrate or pass through lipid bilayer
• make up 25-30% of all encoded proteins and 60% of current drug targets

Question 3

explain peripheral proteins

Answer 3

• located entirely outside of bilayer on either the extracellular or cytoplasmic side
• associated with membrane surface by non-covalent bonds
• easily solubilized

Question 4

explain lipid-anchored membrane proteins

Answer 4

• located outside the membrane (either side) but covalently linked (anchored) to a membrane lipid

Question 5

Integral membrane proteins contain one or more hydrophobic regions embedded into the membrane. What do they function as?

Answer 5

• Transporters: moving ions and solutes across the membrane
• Anchors: binding intra-or extra- cellular components to the membrane
• Receptors: binding ligands to initiate signal transduction pathways
• Enzymes: can catalyze reactions both the intracellular and exoplasmic side of membrane
• Electron Transporters: transfer electrons during photosynthesis and respiration

Question 6

How are integral membrane proteins closely associated with membrane lipids?

Answer 6

• amphipathic (hydrophilic and hydrophobic portions)
• hydrophobic transmembrane domains form van der Waals interactions with the fatty acyl chains of the bilayer
  – this created a seal between proteins and lipids, preserving permeability barrier

Question 7

What is the common secondary structure that spans a membrane?

Answer 7

• hydrophobic a-helix

Question 8

explain a-helix in membrane

Answer 8

• helps by van der Waals interactions of hydrophobic amino acid side chains with lipids
• ionic interactions with polar head groups of lipids

Question 9

What is the a-helix primarily composed of?

Answer 9

• hydrophobic amino acids

Question 10

explain Glycophorin

Answer 10

• 1 of 2 major proteins exposed on the outer surface of human RBCs

Question 11

How do you predict the integral membrane protein from the amino acid sequence?

Answer 11

• Hydropathy plot measures the hydrophobicity of amino acids (determined from their lipid solubility or energy required to transfer them from a nonpolar into an aqueous medium)
• uses a hydropathy index, average based on the hydrophobicity values for each amino acid
  – hydrophobic = +ve, hydrophilic = -ve

Question 12

How many amino acids residues are in a typical membrane?

Answer 12

• typical membrane spanning a-helices are 20-30 amino acid residues long

Question 13

explain the 7 membrane spanning helices of Bacteriorhodopsin

Answer 13

• connected by non-helical loops at the inner and outer face of the membrane
• hydrophobic interactions between nonpolar and the fatty acyl groups of membrane lipids anchor protein
• 7 helices are clustered together and oriented not quite perpendicular to the lipid bilayer and provide a transmembrane proton pathway

Question 14

What is another type of membrane spanning domain? Where are they usually found?

Answer 14

B-barrels
- abundant in outer membrane of gram negative bacteria and the outer membrane of mitochondria and chloroplasts
- many form pores in the membrane allowing the passage of small hydrophilic molecules

Question 15

What amino acids would you expect to line a pore, forming B-barrel?

Answer 15

• inside will have hydrophilic amino acids
• outside will have hydrophobic amino acids

Question 16

How can proteins be attached to the cytosolic side?

Answer 16

• via covalently attached fatty acid chain (acylation - Gly embedded to membrane) or a prenyl group (prenylation - Cys embedded to membrane)

Question 17

What is Farnesyl?

Answer 17

15 carbon phenyl group anchor
- intermediate in the cholesterol biosynthetic pathway
- built from 5 carbon isoprene units

Question 18

What is Geranylgeranyl?

Answer 18

20 carbon prenyl anchor

Question 19

How do proteins attach to the noncytosolic side of the membrane? What is this called?

Answer 19

• by an oligosaccharide linker to the phospholipid phosphatidylinositol
• called a glycosylphosphatidylinositol (GPI) anchor

Question 20

What do GPI anchors attach to?

Answer 20

• attach to proteins in the lumen of the ER
• always on the extracellular face of the plasma membrane
• protein synthesized by rough ER

Question 21

What do Peripheral Membrane Proteins interact/associate with?

Answer 21

• do not interact with the hydrophobic core of the lipid bilayer
• associated with the membrane through interactions with integral membrane proteins or the lipid’s polar head groups

Question 22

How do you remove peripheral membrane proteins? (compared to integral membrane proteins)

Answer 22

• changes in pH or ionic strength

Question 23

explain carbohydrates and glycosylation

Answer 23

• Most plasma membrane proteins are glycosylated.
• The carbohydrate chains face the exoplasmic domain (outside of the membrane).
• Glycolipids are also located in the exoplasmic leaflet of the membrane.

Question 24

How do you solubilize membrane proteins?

Answer 24

• Integral membrane proteins are difficult to isolate because they aggregate in water.
• Amphipathic detergents help to:
  – Solubilize membrane proteins
  – Purify them
  – Reconstitute functional membrane systems
• Types of detergents:
  – Nonionic detergents: Mild, often used for preserving protein function (Triton X-100)
  – Ionic detergents: Harsh, denature proteins, used for complete solubilization (Sodium Dodecylsulfate (SDS))