* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Slide 1
Survey
Document related concepts
Protein phosphorylation wikipedia , lookup
Mechanosensitive channels wikipedia , lookup
Cell nucleus wikipedia , lookup
Cytokinesis wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Membrane potential wikipedia , lookup
Magnesium transporter wikipedia , lookup
Protein moonlighting wikipedia , lookup
Lipid bilayer wikipedia , lookup
Theories of general anaesthetic action wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
Signal transduction wikipedia , lookup
SNARE (protein) wikipedia , lookup
Model lipid bilayer wikipedia , lookup
Cell membrane wikipedia , lookup
List of types of proteins wikipedia , lookup
Transcript
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!