M&R Session 1 (Lecture 1.2)

Question 1

What are the permitted modes of motion of membrane proteins?

Answer 1

1) Conformational change
2) Rotational
3) Lateral

Question 2

What are the restraints on protein mobility?

Answer 2

1) Lipid mediated effects - proteins will generally aggregate to cholesterol poor regions
2) Membrane protein associations
3) Association with extra-membranous proteins e.g. cytoskeleton

Question 3

What are the two types of protein present in membranes?

Answer 3

Peripheral (surface) and Integral (deeply embedded)

Question 4

How is a peripheral membrane protein attached to the membrane, and what can be used to wash these proteins away?

Answer 4

Electrostatic and hydrogen bond interactions. Washed away by changes in pH or using a salt solution (changes in H+ and ionic strength)

Question 5

How is an integral membrane protein attached to the membrane, and what can be used to wash these proteins away?

Answer 5

Interact extensively with hydrophobic regions of bilayer. Require agents e.g. detergents/organic solvents that compete for non-polar interactions is bilayer.

Question 6

What types of AA are present in the lipid region of the bilayer?

Answer 6

Small, hydrophobic and some polar, uncharged e.g. glycine, isoleucine and tyrosine

Question 7

What is the structure of a TM domain?

Answer 7

Alpha helical (3.6aa per turn).

Question 8

Why is asymmetry important for proteins?

Answer 8

Function e.g. correct orientation of Insulin-R is needed for the cell to respond to insulin i.e. ligand binding hydrophilic region is extracellular and not facing internally.

Question 9

How is an erythrocyte ghost membrane formed?

Answer 9

Osmotic haemolysis releasing cytoplasmic components

Question 10

Which molecular technique is used to analyse ghost membranes and what is revealed?

Answer 10

SDS-PAGE

Over 10 major proteins (bands) with major ones being numbered (heaviest to lightest).

Question 11

If most of the proteins in the erythrocyte membrane are washed away with salt/changes in pH and are susceptible to proteolysis when the cytoplasmic face is exposed, what can be said about these proteins and their location?

Answer 11

Most are peripheral proteins located on the cytoplasmic side of the membrane.

Question 12

Protein bands 3 and 7 are washed away with detergents. What does this indicate?

Answer 12

They are deeply embedded, integral proteins.

Question 13

The erythrocyte cytoskeleton is a network composed of which two proteins?

Answer 13

Spectrin and Actin

Question 14

Describe the structure of Spectrin and how it is formed.

Answer 14

Long, floppy, rod like molecule. Alpha and Beta subunits wind together to form an antiparallel heterodimer. 2 heterodimers associate head-to-head to form a heterotetramer of A2B2.

Question 15

What does actin do in the RBC cytoskeleton? What else is present in these ‘crosslinks’?

Answer 15

Crosslinks spectrin molecules along with band 4.1 and adducin.

Question 16

What attaches the spectrin-actin network to the membrane? Which specific proteins does it attach to?

Answer 16

Adaptor proteins : Band 4.1 and Ankyrin (4.9)

Attaches to Band 3 and Glycophorin A

Question 17

What does this attachement do to the mobility of the integral proteins?

Answer 17

Reduces lateral mobility.

Question 18

What is the abnormality in Hereditary Spherocytosis and the inheritance pattern?

Answer 18

One allele of spectrin is not expressed leading to low levels of spectrin.

75% autosomal dominant, few recessive.

Question 19

What is the appearance of RBCs in HS? What does this abnormal appearance cause?

Answer 19

RBCs round up (more spherical), less resistant to lysis.
Cleared by the spleen (due to defective shape and reduced life span)

Leads to haemolytic anaemia (low Hb), splenomegaly, jaundice, gallstones (increased bilirubin) and low levels of HbA1c.

Question 20

What is the abnormality in Hereditary Elliptocytosis and the inheritance pattern?

Answer 20

Defective spectrin that is unable to form heterotetramers. Autosomal dominant (99%)

Question 21

Where is HE most prevalent and why?

Answer 21

Endemic to Africa as it provides resistance to malaria.

Question 22

What is the appearance of RBCs in HE? What does this abnormal appearance cause?

Answer 22

Fragile elliptoid cells (rugby ball shaped) affecting the deformability of cells. Reduced life span so cleared byt spleen.

Like HS, the patient would have haemolytic anaemia and with splenomegaly and jaundice.

Question 23

Describe the process of membrane protein formation.

Answer 23

1) Signal sequence of primary sequence, at the N terminus.
2) Recognised by SRP which halts translation and brings ribosome-mRNA-protein complex to the ER surface.
3) Interaction with SRP-R and release of SRP.
4) Signal sequence is cleaved by signal peptidases.
4) Translation continues with transcolon open to allow protein to enter the ER lumen.
5) For a membrane protein, a stop transfer sequence (hydrophobic stretch of AAs 18-20 in length) in alpha helical arrangement arrests translation.
6) This allows the protein to be embedded into the membrane with its N termini in the lumen and C termini extracellular.

Question 24

What is the main effector of determining the oreintation of the N and C termini?

Answer 24

Location of start-transfer sequences within the primary sequence.

Question 25

What is a start-transfer sequence?

Answer 25

Stretch of AA that are positively charged being at either the N or C termini.

Question 26

Describe how multiple transmembrane regions are created?

Answer 26

Created cytoplasmically and inserted with the help of heat shock proteins and BiPs.