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Tutorial 4
Introduction - membranes
What are membranes?
Membranes are barriers that define compartments
• They are made up of a lipid bilayer
Membrane Proteins
• They carry out many functions
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.
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?
Average Hydropathy Index
Hydropathy Plots
0
50
100
150
200
Amino Acid Number
250
Hydrophobicity
Hydropathy plot for Glycophorin A
0
How many times does this protein span the membrane?
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
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.
A
A
A
A
B
C
D
E
Gel 1
All
OUTSIDE
INSIDE
B
C
E
Gel 2
Carbohydrates
D
E
Gel 3
outside
B
D
Gel 4
inside
Gel 5
Free proteins
• 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!
0
What can we
conclude
from these
results?
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.
CELL FUSION
Figure 11-35 (p.366)
- 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
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
NEXT TUTORIAL:
STRUCTURE AND FUNCTION OF
MITOCHONDRIA AND
CHLOROPLAST!