Cell Biology 4033 Lecture #24 p491-512 Membrane Proteins and Transport Chapter 10;

Chapter 11 lecture #24 p. 507-512

Membranes: "Where the action is"

Discerning orientation of membrane proteins; label right-side-out, inverse, digest transmambrane, bind antibodies, etc.

Spectrin(25%), glycophorin(numerous, unknown function), BandIII (HCO3into RBC) =60%

spectrin-ankyrin-bandIII +band 4.1(other participants) link cytoskeletin to RBC membrane (fig 10-26)

"P" (protoplasmic) and "E"(extracellular) freeze-fracture faces of membrane.

Bacteriorhodopsin of Halobacterium halobium (proton-motive force generator) 7 alpha helices

"homologous" proteins. Similar structures, very different functions

Porins = beta-barrels in outer membrane of G-. Selective transport.

Fluorescent antibodies + rhodamine or fluorescene to support immunon model (patching, capping, endocytosis)

Junctions restrict proteins to "domains". Aggregates, tethering(in or out), interact with cells.

Glycocalyx, sugars, lectins (use of ConA [mannose binding]in protein purification )

Sperm-egg interaction, blood clotting, lymphocyte recirculation, inflammation all involve lectins

Margination and diapedesis involve selectins (immunoglobulin superfamily)

Chapter 11 lecture #24 p. 507-512

1/5 of E. coli genes = transport. Problem: selective crossing of fat layer.

1.Carrier proteins (linkable to energy source for active transport). Permeases, transporters

2.Channel proteins (facilitated diffusion[concentration, membrane potential]; hydrophilic pores)

Electrochemical gradient established= energy to drive all sorts of processes.

K⁺, H⁺ higher inside; Na+,Mg⁺², Ca⁺², Cl^- higher outside. Usually net negative inside.

Small, nonpolar, hydrophobic, uncharged = cross fatty layer. H₂O Billion X than Na⁺, K⁺

recombinant DNA: Sequence DNA, get a.a. sequence, hydropathy plot shows transmembrane patches.

Antibody (specific binding)-based examination of regions inside and outside membrane.

Site-specific mRNA mutagenesis, inject into cell= evaluate changes. Critical a.a. identified.

Probe to identify homologous DNA sequences. Transporters = very few, related families, although many proteins in each family.

Ionophores: mobile carriers (valinomycin), channel formers (gramicidin). Biological warfare? No; simply create a zone of clearing in the immediate vicinity