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. Is the exposed surface made of polar or nonpolar groups?
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 Amino Acid Number 50 100 15050
100
150
200
250
Amino Acid Number

7. Hydropathy plot for Glycophorin A
Hydrophobicity
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
inside
Free proteins
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?

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
Figure (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

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. STRUCTURE AND FUNCTION OF MITOCHONDRIA AND CHLOROPLAST!
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STRUCTURE AND FUNCTION OF MITOCHONDRIA AND CHLOROPLAST!