Now playing Conrad Herwig (Rutgers Artist in Residence) at the Blue Note

1. Now playing Conrad Herwig(Rutgers Artist in Residence)at the Blue Note

2. Membranes and Protein TargetingCharles MartinB323 Nelson Labs

3. Membranes organize cells into functionally distinct compartments • Each type of membrane has a unique function and unique protein and lipid components • The interior (lumen) of each compartment has a unique chemical composition • Membranes control the composition of the compartments by controlling movement of molecules across the membrane

4. The basic structural unit of biological membranes is a lipid bilayer

7. Phospholipids are the primary bilayer forming lipids of cell membranes • Phospholipids contain fatty acids linked to glycerol by ester bonds at carbons 1 and 2 • Fatty acyl chains can be saturated or unsaturated • An alcohol headgroup is linked to glycerol at carbon 3 by phosphodiester bond.

8. Headgroups of membrane phospholipids • Choline, ethanolamine are the most abundant PL classes. Headgroup has no net charge • Serine and inositol headgroups have net negative charges

9. Phospholipids are amphipathic molecules • The glycerol and headgroup moieties are hydrophilic - readily associate with water • The fatty acyl part of the molecule is hydrophobic – disrupt the ordered structure of water

10. Most naturally occurring phospholipids form bilayers when they are dispersed in water • Polar headgroups and glycerol backbone are associated with surrounding water • The hydrophobic fatty acyl chains are confined to the interior out of contact with aqueous environment

11. Detergents and lysoglycerolipids form micelles • Determined by the shape of the molecule • Single fatty acid in lyso-PL or hydrocarbon chain in detergents creates a conical molecule that has too high a rate of curvature to form planar bilayer

12. Bilayers abhor free ends • Pure phospholipid bilayers spontaneously seal to form closed structures

13. Cell membranes are asymmetric • Cellular membranes have a cytosolic face (exposed to the cytosol) and an exoplasmic face (directed away from the cytosol) • Organelles with two membranes, the exoplasmic surface faces the lumen between the membranes

14. Each closed compartment has two faces Each leaflet of a membrane has a different lipid and protein composition

16. Membrane lipid bilayers are liquid crystals that behave as 2-dimensional fluids • Below the phase transition temperature fatty acyl chains are in a gel-like (crystalline) state • Above the phase transition temperature, fatty acyl chains are in rapid motion

17. 6- 20 hours for flip-flop • Phospholipids can rapidly diffuse along the plane of the membrane • Nearest neighbor replacement rate is ~10-8/sec • Flip-flop is a rare process – • leaflet exchange rate is 6 - > 20 h 10-8 sec

18. Van der Waals interactions between fatty acyl chains are the main determinants of acyl chain mobility

19. van der Waals forces are strongly dependent on interatomic distance

20. Double bonds reduce the number of potential van der Walls interactions between fatty acyl chains

21. Cholesterol is an amphipathic steroid that is abundant in plasma membranes

22. Steroid nucleus is planar hydrophobic molecule • Hydroxyl group of cholesterol interacts with water

23. Cholesterol can pack with phospholipids in a 1:1 ratio

24. The “Fluidity” of a Lipid Bilayer Is Determined by Its Composition • Short chain fatty acyl groups tend to increase lateral mobility • Unsaturated fatty acids tend to increase fluidity • Cholesterol and other sterols tend to impede fatty acid mobility (act as a fluidity buffer)

25. Sphingolipids and glycolipids are found on the surface of all plasma membranes

26. Very Long Chain Fatty Acid • Sphingolipids are derived from serine, not glycerol • Long chain base (sphingosine) linked to very long chain (usually C26 – C28) fatty acid by N-acyl bond

27. Sphingolipids and cholesterol segregate into “raft” domains on the plasma membrane • One type of cholesterol /sphingolipid enriched microdomains are found in caveolae – small pits on cell surface • Caveolae appear to function : • in certain types of endocytosis, • as organizing centers for signaling molecules • in mechanotransduction (monitor blood flow over endothelial cell surface) Dynamin immunogold 5 nm Coated pit caveolae

28. Caveolin is the major protein in caveolae • Can bind to cell surface receptors • NOS, Ras, PKC a & b, EGFR, PDGFR • Caveolin interacts as negative regulator with signaling molecules through 20 aa caveolin scaffolding domain • Cholera toxin – • import is blocked in cells with mutant caveolin

29. Membrane proteins can be associated with the lipid bilayer in different ways

31. The polypeptide chains of most transmembrane proteins cross the bilayer in an a-helical conformationA typical transmembrane a-helix consists of ~ 20-25 hydrophobic amino acids

32. Glycophorin monomers span the red blood cell membrane with a single transmembrane -helix

33. The TM a-helices of two glycophorin membrane spanning regions associate as a coiled-coil structure forming a dimer

34. Porins are pore-forming proteins that span the bilayer as a b-barrel • Rhodobacter porin monomer (a trimer in membrane) • 16 antiparallel b-sheets • Hydrophobic side chains exposed to bilayer • Hydrophilic residues exposed to pore

35. Intrinsic membrane proteins can pass through the bilayer many times Muscle Ca++ ATPase Mammalian glucose symporter

37. Other membrane proteins are attached to the bilayer by covalently attached lipids

38. Myristoylated proteins contain a covalently attached 14-carbon fatty acid at the N-terminus of the protein Myristoylation occurs in initial phases of protein synthesis

39. Prenyl and palmitoyl groups are attached to cysteine residues via a thioether linkage • Prenyl groups are unsaturated intermediates of sterol synthesis • Palmitic acid is a 16 carbon saturated fatty acid • These protein modifications occur after the protein is synthesized

40. Glycerophosphatidylinositol serves as a covalently bound phospholipid anchor for certain cell surface proteins • GPI proteins are found on cell surface • Lipid modification occurs after protein is inserted through ER bilayer

41. Some protein domains can attach or release from membranes by changing their conformation

42. C2 domains can be found on many different types of proteins

43. C2 domains typically bind 3 Calcium atoms • 2, 4-stranded b-sheets • 5 conserved Asp residues and one serine bind 3 calcium ions at top • (+) Ca++ binds anionic PL • (-) Ca++, release from lipid surface

44. PLA-2 C2 domains change their surface potential on binding calcium.Murray &Honig Cell, 2002 Sytl-C2A Sytl-C2A – 25 mV EP contour Ca binding region

45. Pleckstrin Homology (PH) Domains target proteins to membranes by binding to specific phosphoinositol phospholipids • PH domains are found in over 250 proteins in human genome • Bind to specific phosphorylated forms of phosphatidyl inositol

46. pleckstrin domain 7 –stranded b-sandwich closed on one side by an a - helix

47. Single molecule fluorescence detection shows that pleckstrin domains can bind to immobilized patches of membrane • Myosin X – • dimeric molecular motor with 3 pleckstrin homology domains • Binds to inner surface of plasma membrane in stimulated cells to generate force by binding to actin molecules in cell ruffling

48. Detection of single molecules of eGFP-PH123 molecules in the lamella of a living mouse myoblast under time-lapse recording Mashanov, G. I. et al. J. Biol. Chem. 2004;279:15274-15280

49. Average residency rate of myosin X eGFP is 20 sec • Either bound to cytoskeleton or to corralled lipid environment

50. Phosphatidyl inositols can act as molecular switches that recruit proteins to different membrane surfaces.