Introduction - membranes

1
Tutorial 4
Introduction - membranes
2
What are membranes?
Membranes are barriers that define compartments

• They are made up of a lipid bilayer

3
Membrane Proteins

• They carry out many functions

4
Membrane Proteins
Integral - firmly anchored into the membrane by
hydrophobic interactions with the hydrophobic
portion of the bilayer.
Transmembrane proteins - extend through membrane
Lipid anchored proteins - have covalently
attached lipid molecules that anchor the protein
into the bilayer
Peripheral - attached to surface of membrane and
to the exposed parts of integral proteins. Held
in place by polar interactions primarily.
5
Freeze Fracture

• A technique used to visualize protein
  distribution in a membrane

This figure was modified from Bloom and Fawcett,
A Textbook of Histology, Chapman and Hall, N.Y.,
Twelfth Edition, 1994, Figure 1-3
Is the exposed surface made of polar or nonpolar
groups?
6
Hydropathy Plots
Average Hydropathy Index
0
50
100
150
200
250
Amino Acid Number
7
Hydropathy plot for Glycophorin A
How many times does this protein span the
membrane?
8
Membrane Experiments 1

• Gel Electrophoresis can be used to identify the
  types of proteins found within a membrane.
• Different stains can tell you different things
  about the membrane proteins
• Different cell preparations can allow you to view
  internal vs external proteins

9
Experiment

• Gel 1 stain ALL proteins with Coomassie Blue
• Gel 2 remove Coomassie Blue and stain with a
  carbohydrate stain.

• Gel 3 Treat fresh cells with a fluorescent
  marker that binds proteins on the outside of
  cells.
• Gel 4 Using fresh cells, make inside-out
  vesicles. Label inside out vesicles using a
  fluorescent marker.
• Gel 5 Separate free proteins from the
  membranes.

10
A
A
A
A
B
B
B
C
C
D
D
D
E
E
E
Gel 1
Gel 2
Gel 3
Gel 4
Gel 5
All
Carbohydrates
outside
Free proteins
inside
OUTSIDE
INSIDE
11

• Polypeptide A
• Found both on inside and outside of membrane.
  How many times does it span the membrane???
• We can solve this with a hydropathy plot!

What can we conclude from these results?
0
12
Membrane Experiments 2

• MEMBRANE FLUIDITY can be observed using
  fluorescent dyes.
• Different dyes can label different proteins
• Proteins can move laterally quite easily and this
  can be observed
• Note only certain proteins are able to flip to
  the other membrane bilayer via a flippase and
  this is quite specialized.

13
CELL FUSION
- Surface proteins of cultured cells are labeled
with antibodies coupled to fluorescent dyes (red
and green). - The "red" and "green" cells are
then mixed and can fuse. - In time, labeled
proteins from each cell mix showing membrane
fluidity
14
FRAP fluorescence recovery after photobleaching
An experiment to demonstrate fluidity of membrane
components. The green indicates green
fluorescence
A the location of the spot to be
photobleached B the spot after being
bleached by radiation C, D disappearance of the
spot as time goes on due to the fluidity of the
membrane
15
NEXT TUTORIAL

• STRUCTURE AND FUNCTION OF MITOCHONDRIA AND
  CHLOROPLAST!