membranes and transport

Question 1

what is a hydropathy plot used for? why is this useful?

Answer 1

to identify a region of hydrophobic residues in a sequence. this is useful as a TM helix has a high hydrophobic score. can help identify TM helices

Question 2

using a hydropathy plot, when is a sequence considered to be part of a TM helix?

Answer 2

when there is a region of 20 or more residues of high hydropathy

Question 3

what is meant by the Von Heijne positive inside rule?

Answer 3

positively charged lysine, histidine and arginine resides of membrane proteins are located more on the cytoplasmic face of the membrane

Question 4

what is the usefulness of the Von Heijne positive inside rule?

Answer 4

allows prediction of orientation of helices

Question 5

what is a B-barrel?

Answer 5

20 or more transmembrane segments from B-sheets that line a cylinder

Question 6

give some examples of membrane proteins that are B-barrels

Answer 6

FepA, OmpLA, Maltoporin

Question 7

what are porins?

Answer 7

membrane proteins with a hydrophobic exterior and hydrophilic core, usually involved in transport and located in the membrane

Question 8

what do membrane anchors do?

Answer 8

attached lipid provides a hydrophobic anchor that inserts into the lipid bilayer and holds the protein at the membrane surface

Question 9

which techniques can be used in biophysical studies to investigate membrane structure?

Answer 9

• X-ray crystallography
• NMR spectroscopy
• Electron microscopy

Question 10

why are crystallographic studies into membrane proteins difficult?

Answer 10

• membrane proteins are difficult to crystallise

- difficult to overexpress and purify membrane proteins

Question 11

why is the requirement for controlled expression and reconstitution of membrane proteins an issue for biophysical studies?

Answer 11

amphipathic molecules aggregate in aqueous solutions, therefore, the native conformation is distorted

Question 12

how can the use of GFP solve the problems associated with biophysical studies of membrane proteins?

Answer 12

verify the C terminus of the protein of interest is cytoplasmic and tag with GFP, this way you can identify the conformation

Question 13

aside from the use of GFP, what can be used to help perform biophysical studies into membrane proteins?

Answer 13

• use of detergent to solubilise the membrane, prevents aggregation
  e. g. polyacrylate based amphiphols and lipopeptides
• chemical modifications
• mutations to increase thermostability
• solubilising tags

Question 14

what is the patch clamp technique used for?

Answer 14

measuring channel activity. can provide a measure of:

• how fast the channel opens and closes
• how frequently the channel opens and how long it stays open
• effect of membrane potential on channel function

Question 15

how is the patch clamp technique used?

Answer 15

• high resistance seal is formed between the pipette and and a small patch of plasma membrane
• breaking of the membrane patch by increased suction produces a low resistance pathway between the pipette and interior of the cell
• pipette is pulled away from the cell and a piece of plasma membrane with its cytosolic side facing the medium is monitored by the patch pipette

Question 16

what is meant by the ball and chain model?

Answer 16

rapid inactivation via occlusion of the pore.

• when the channel is closed, the ball rotates freely in aqueous solution
• when the channel opens, the ball quickly finds a complementary site in the open pore and occludes it

Question 17

how does the structure of calmodulin allow it to regulate a large variety of proteins?

Answer 17

it is flexible, with a generic shape for the non-polar grooves in its binding site

Question 18

outline the mechanism for CaM

Answer 18

• 4 EF hand motifs as the binding site for calcium

- the alpha helices of the EF hand motif permits a change in the binding affinity for the target protein

Question 19

how can trafficking regulate channel proteins?

Answer 19

can occur via -

• translocation and insertion
• endocytosis e.g. removal from the membrane
• recycling

Question 20

how can defective transport proteins result in disease?

Answer 20

long-QT has many variants many of these are associated with ion channels. action potentials integrate the activities of several ion channels working in concert