Tutorial 4

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. 0 Hydrophobicity 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 carbohydratestain. • 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. Figure 11-35 (p.366) 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!