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

Integral - firmly anchored into the membrane by hydrophobic interactions with the hydrophobic portion of the bilayer.

Membrane Proteins

Peripheral - attached to surface of membrane and to the exposed parts of integral proteins. Held in place by polar interactions primarily.

Transmembrane proteins - extend through membrane

Lipid anchored proteins - have covalently attached lipid molecules that anchor the protein into the bilayer

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?

Amino Acid Number

50 100 150 200 250

0

Ave

rage

Hyd

ropa

thy

Inde

xHydropathy Plots

Hyd

roph

obic

ity

0

Hydropathy plot for Glycophorin A

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

DE

Gel 3

A

BC

Gel 4

B

D

Gel 5

A

BCDE

A

E

Gel 1 Gel 2All Carbohydrates outside inside Free proteins

OUTSIDE

INSIDE

• 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.

- 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

CELL FUSION

Figure 11-35 (p.366)

An experiment to demonstrate fluidity of membrane components. The green indicates green fluorescence

FRAP – fluorescence recovery after photobleaching

A = the location of the spot to be photobleached B = the spot after being bleached by radiationC, 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!