Membrane Proteins and Transport

1. Membrane Proteins and Transport BL4010 11.28.06

2. Outline • Passive Diffusion • Facilitated Diffusion • Active Transport • Transport Driven by ATP, light, etc. • Specialized Membrane Pores • Ionophore Antibiotics

3. http://www.stolaf.edu/people/giannini/flashanimat/transport/osmosis.swfhttp://www.stolaf.edu/people/giannini/flashanimat/transport/osmosis.swf

4. Osmotic potential

5. Electric Potential

6. Passive Diffusion No special proteins needed • Transported species simply moves down its concentration gradient - from high [c] to low [c] • High permeability coefficients usually mean that passive diffusion is not the whole story

8. Facilitated Diffusion G negative, but proteins assist • Solutes only move in the thermodynamically favored direction • But proteins may "facilitate" transport, increasing the rates of transport • Two important distinguising features: • solute flows only in the favored direction • transport displays saturation kinetics

11. Facilitated DiffusionBeware the cartoon world! • Uniport • http://www.stolaf.edu/people/giannini/flashanimat/transport/caryprot.swf • Gated channel • http://www.stolaf.edu/people/giannini/flashanimat/transport/channel.swf • Symport • http://www.stolaf.edu/people/giannini/flashanimat/transport/symport2.swf

12. Active Transport Systems Energy input drives transport • Some transport must occur such that solutes flow against thermodynamic potential • Energy input drives transport • Energy source and transport machinery are "coupled" • Energy source may be ATP, light or a concentration gradient

13. Trp (red) and Tyr (gold) at the interface

14. Secondary Active Transport Transport processes driven by ion gradients • Many amino acids and sugars are accumulated by cells in transport processes driven by ion gradients

15. Secondary Active Transport • Symport - ion and the amino acid or sugar are transported in the same direction across the membrane • Antiport - ion and transported species move in opposite directions • Several examples are described in Table 10.2

16. Porins Found both in Gram-negative bacteria and in mitochondrial outer membrane • Porins are pore-forming proteins - 30-50 kD • General or specific - exclusion limits 600-6000 • Most arrange in membrane as trimers • High homology between various porins • Porin from Rhodobacter capsulatus has 16-stranded beta barrel that traverses the membrane to form the pore (with eyelet!)

19. Why Beta Sheets? for membrane proteins?? • Genetic economy • Alpha helix requires 21-25 residues per transmembrane strand • Beta-strand requires only 9-11 residues per transmembrane strand • Thus, with beta strands , a given amount of genetic material can make a larger number of trans-membrane segments

20. The Pore-Forming Toxins • Lethal molecules produced by many organisms • They insert themselves into the host cell plasma membrane • They kill by collapsing ion gradients, facilitating entry by toxic agents, or introducing a harmful catalytic activity

21. Colicins • Produced by E. coli • Inhibit growth of other bacteria (even other strains of E. coli) • Single colicin molecule can kill a host! • Three domains: translocation (T), receptor-binding (R), and channel-forming (C)

22. colicin Ia, 210 Å spans periplasmic space of gram negative bacteria. blue=receptor binding (o.m.) violet=C-domain forms channel on i.m. green=hydrophobic red=translocon

23. Clues to Channel Formation • C-domain: 10-helix bundle, with H8 and H9 forming a hydrophobic hairpin • Other helices amphipathic (Fig. 10.30) • H8 and H9 insert, with others splayed on the membrane surface • A transmembrane potential causes the amphipathic helices to insert!

25. Other Pore-Forming Toxins • Delta endotoxin also possesses a helix-bundle and may work the same way • There are other mechanisms at work in other toxins • Hemolysin from Staphylococcus aureus forms a symmetrical pore • Aerolysin may form a heptameric pore - with each monomer providing 3 beta strands to a membrane-spanning barrel

26. B.t. toxin (left) and diptheria toxin

27. Hemolysin allows Ca2+ influx, kills blood cells (Staph. aureus)

28. Amphiphilic Helicesform Transmembrane Ion Channels • Many natural peptides form oligomeric transmembrane channels • The peptides form amphiphilic -helices • Aggregates of these helices form channels that have a hydrophobic surface and a polar center • Melittin (bee venom), magainins (frogs) and cecropin (from cecropia moths) are examples

29. Melittin • Bee venom • 26 amino acids • monomer in membrane until potential is applied then tetrameric chlorid channel • causes pores to form in nocireceptors (pain) • stimulates the nerve, pain response, resetting of nerve causes deoligomerization but re-established with next resting potential

30. Amphipathic Helices • Melittin - bee venom toxin - 26 residues • Cecropin A - cecropia moths - 37 residues • Magainin 2 amide - frogs - 23 residues • See Figure 10.35 to appreciate helical wheel presentation of the amphipathic helix

31. The Magainin Peptides • Discovered by Michael Zasloff • He noticed that incisions on Xenopus laevis (African clawed frog) healed without infection, even in bacteria-filled aquarium water • He deduced that the frogs produced a substance that protected them from infection!

33. The Cecropins • Produced by Hyalophora cecropia Induced when the moth is challenged by bacterial infections • These peptides are thought to form helical aggregates in membranes, creating an ion channel in the center of the aggregate

35. Gap Junctions Vital connections for animal cells • Provide metabolic connections • Provide a means of chemical transfer • Provide a means of communication • Permit large number of cells to act in synchrony

36. Gap Junctions • Hexameric arrays of a single 32 kD protein • Subunits are tilted with respect to central axis • Pore in center can be opened or closed by the tilting of the subunits, e.g. as response to stress

39. Ionophore Antibiotics Mobile carrier or pore (channel) • How to distinguish? Temperature! • Pores will not be greatly affected by temperature, so transport rates are approximately constant over large temperature ranges • Carriers depend on the fluidity of the membrane, so transport rates are highly sensitive to temperature, especially near the phase transition of the membrane lipids

41. Valinomycin A classic mobile carrier • A depsipeptide - a molecule with both peptide and ester bonds • Valinomycin is a dodecadepsipeptide • The structure places several carbonyl oxygens in the center of the ring structure • Potassium and other ions coordinate the oxygens • Valinomycin-potassium complex diffuses freely and rapid across membranes

43. Selectivity of Valinomycin Why? • K + and Rb + bind tightly, but affinities for Na + and Li + are about a thousand-fold lower • Radius of the ions is one consideration • Hydration is another it "costs more" energetically to desolvate Na+ and Li+ than K+

44. Gramicidin A classic channel ionophore • Linear 15-residue peptide - alternating D & L • Structure in organic solvents is double helical • Structure in water is end-to-end helical dimer • Unusual helix - 6.3 residues per turn with a central hole - 0.4 nm or 4 A diameter • Ions migrate through the central pore

48. The Sodium Pump aka Na+/K+-ATPase • Large protein - 120 kD and 35 kD subunits • Maintains intracellular low Na+ and high K+ • Crucial for all organs, but especially for neural tissue and the brain • ATP hydrolysis drives 3Na+ out and 2K+ in • Alpha subunit has ten transmembrane helices with large cytoplasmic domain

49. Sequence alignment

50. The Na+/K+ ATPase • ATP hydrolysis drives 3Na+ out and 2K+ in