Medical Biochemistry

1. Medical Biochemistry Membranes: Bilayer Properties, Transport Lecture 71

2. Membrane function • Serve as barriers to separate contents of cell from external environment or contents of organelles form remainder of the cell • Proteins in cell membrane have many functions • transport of substances across the membrane • enzymes that catalyze biochemical reactions • receptors on exterior surface that bind external ligands (e.g., hormones, growth factors) • mediators that aid ligand-receptor complex in triggering sequence of events (second messengers that alter metabolism are produced inside the cell)

3. Plasma membrane has selective permeabilities • Channels and pumps • for ions and substrates • Specific receptors • for signals (e.g., hormones) • Exchange materials with extracellular environment • exocytosis and endocytosis

4. Membranes form specialized compartments • Organelles with specialized functions • e.g., mitochondria, ER, Golgi complex • Localize enzymes • Excitation-response coupling • Energy Transduction • photosynthesis, oxidativephosphorylation

5. Internal Water Is Compartmentalized • Intracellular Fluid (2/3 of total water) • rich in K+ and Mg2+, phosphate major anion • protein higher • Extracellular Fluid (1/3 of total water) • high Na+ and Ca+, chloride major anion • glucose higher

6. Composition of membranes varies within and between cells • Major lipids in mammalian membranes • Phospholipids • Glycosphingolipids • Cholesterol

7. Phospholipids - two major classes 1. phosphoglycerides are more common • glycerol backbone • two fatty acids in ester linkage • usually even-numbered carbons (C16, C18) • unbranched, either saturated or unsaturated • C18 or 20:4,5,8,11,14 • phosphorylated alcohol • phosphatidic acid (1,2-diacylglycerol 3-phosphate) is simplest -- key intermediate in formation of all other phospholipids

8. Phospholipids - two major classes 2. sphingomyelins • sphingosine backbone (rather than glycerol) • fatty acid attached by amide linkage • primary hydroxyl group of sphingosine esterified to phosphocholine • prominent in myelin sheaths

9. Glycosphingolipids • sugar-containing lipids • e.g., cerebrosides and gangliosides • also derived from sphingosine • differ from sphingomyelin in group attached to primary hydroxyl group of sphingosine • sphingomyelin - phosphocholine • cerebroside - single hexose (glucose or galactose) • ganglioside - chain of 3 or more sugars (at least one is sialic acid)

10. Sterols • most common sterol cholesterol • almost exclusively in plasma membrane • lesser amounts in mitochondria, Golgi, nuclear membranes • generally more abundant toward outside of plasma membrane • intercalates among phospholipids of membrane with its hydroxyl group at aqueous interface and remainder of molecule within leaflet

11. Membrane lipids are amphipathic • Contain both hydrophobic and hydrophilic regions (like detergents) • polar head group • nonpolar tails • Saturated fatty acids - straight tails • Unsaturated fatty acids (generally cis) - kinked tails

12. What is the effect of unsaturated fatty acids?

13. What is the effect of unsaturated fatty acids? • as more kinks added, membrane becomes less tightly packed, more fluid

14. Membrane lipids form bilayers • Amphipathic phospholipids have two regions with incompatible solubilities • in aqueous solvent, organize into thermodynamically favorable form (e.g., micelle)

15. Membrane lipids form bilayers • Bimolecular layer (bilayer) can also satisfy thermodynamic requirement of amphipathic molecule • only ends or edges of bilayer sheet exposed to unfavorable environment • can eliminate by folding sheet back upon itself to form enclosed vesicle with no edges. • Closed bilayer is essential property of membrane • impermeable to most water-soluble molecules

16. Lipid-soluble materials • Gases (oxygen, CO2, nitrogen) • little interaction with solvents, readily diffuse through hydrophobic regions of membrane • Lipid-derived molecules (e.g., steroid hormones) • readily transverse bilayer • Organic nonelectrolyte molecules • diffusion dependent upon oil-water partition coefficients (the greater lipid solubility, the greater its diffusion rate across membrane)

17. Non-lipid-soluble molecules • Proteins are also amphipathic molecules • inserted into lipid bilayer • form channels for movement of ions and small molecules • serve as transporters for larger molecules

18. Non-lipid-soluble molecules • Side chains determine hydrophobic nature • 6 strongly hydrophobic side chains, few weakly hydrophobic, remainder hydrophilic • amphipathic proteins have hydrophobic region transversing bilayer and hydrophilic regions protruding inside and outside of membrane • protein content varies with membrane • enzymes, transport proteins, receptors

19. Membranes and components are dynamic structures • Lipids and proteins in membranes turn over • different lipids and proteins have individual turnover rates, may vary widely • membrane may turn over more rapidly than any of its constituents

20. Membranes Are Asymmetric Structures • Irregular distribution of proteins within membrane • External location of carbohydrates attached to membrane proteins • Regional asymmetries • villous border of mucosal cells • gap junctions, tight junctions,synapses

21. Membranes Are Asymmetric Structures • Phospholipid asymmetry • choline-containing phospholipids located mainly in outer leaflet • phosphatidylcholine, sphingomyelin • aminophospholipids preferentially located in inner layer • phosphatidylserine, phosphatidylethanolamine • cholesterol generally present in larger amounts on the outside

22. Membranes Are Asymmetric Structures • Must be limited transverse mobility (flip-flop) • half-life of asymmetry in synthetic bilayers is several weeks • enzymes for phospholipid synthesis are located on cytoplasmic side of microsomal membranes • flippases • phospholipid exchange proteins

23. Integral and peripheral proteins • Integral membrane proteins • interact with phospholipids, require detergents for solubilization • usually globular, amphipathic • may span bilayer many times • asymmetrically distributed across bilayer • orientation determined during insertion in bilayer

24. Integral and peripheral proteins • Peripheral proteins • do not interact directly with phospholipids • do not require detergent for release • weakly bound to hydrophilic regions of specific integral proteins

25. Integral and peripheral proteins • e.g., ankyrin, bound to integral protein “band 3” of erythrocyte membrane • spectrin, a cytoskeletal structure within erythrocyte, bound to ankyrin • plays important role in maintenance of biconcave shape of erythrocyte

26. Artificial membranes model membrane function • Mixtures of one or more phospholipids treated (e.g., sonication) to form spherical vesicles  liposomes • can control lipid content to examine effects of lipid composition on certain functions • purified membrane proteins can be incorporated into these vesicles to access factors required for function • environment can be controlled and varied (e.g., ion concentrations) • can be made to entrap compounds inside (e.g., drugs, isolated genes) for drug delivery, gene therapy

27. Fluid mosaic model • Singer and Nicolson (1972) • icebergs (membrane proteins) floating in a sea of predominantly phospholipid molecules • translational diffusion - integral proteins and phospholipids can move within the plane of the membrane

28. Fluid mosaic model • phase changes (fluidity) of membrane are dependent upon lipid composition • hydrophobic chains of fatty acids can be highly ordered  rigid structure • with  temperature, side chains undergo transition from ordered state (gel-like or crystalline phase) to disordered (liquid-like or fluid) phase • transition temperature (Tm) • longer, more saturated fatty acid chains interact more strongly, cause higher Tm • unsaturated chains tend to  fluidity,  compactness

29. Fluid mosaic model • Cholesterol modifies fluidity of membranes • At temperatures below Tm it interferes with the interaction of hydrocarbon tails of fatty acids and increases fluidity • At temperatures above Tm it limits disorder because it is more rigid than tails of fatty acids and cannot move in membrane to same extent, thus limits fluidity • At high cholesterol:phospholipid ratios, transition temperatures are abolished

30. Fluid mosaic model • Fluidity significantly affects membrane functions • As membrane fluidity , so does permeability to water and other small hydrophilic molecules • Lateral mobility of integral proteins increases • If active site of integral protein resides exclusively in hydrophilic regions, changing fluidity probably has little effect on activity • If protein involved in transport, with transport components span membrane, lipid phase effects may significantly alter transport rate. • EXAMPLE: Insulin receptor - As concentration of unsaturated fatty acids in membrane increased (grow in unsaturated. fatty acid rich medium), fluidity increases, receptor binds more insulin

31. Fluid mosaic model • Some protein-protein interactions within plane of membrane can restrict mobility of integral proteins

32. Asymmetry of proteins and lipids maintained during membrane assembly • Fusion of a vesicle with the plasma membrane preserves the orientation of any integral proteins embedded in the vesicle bilayer

33. Signal Sequences Target Many Proteins • Many proteins carry signals that target them to their destination • Major sorting decision - synthesis on free or membrane-bound polyribosomes • cytosolic branch • no signal peptide, delivered tocytosol • can be directed to mitochondria, nuclei, peroxisomes by specific signals

34. Signal Sequences Target Many Proteins • rough ER branch (Secretory or exocytotic pathway) • contain signal peptide • many destined for various membranes (ER, Golgi, lysosomes, and plasma membrane) and for secretion • certain proteins sorted in Golgi for delivery to lysosomes • proteins destined for secretion carried in secretory vesicles • regulated secretion (secretory vesicles) • constitutive secretion (transport vesicles)

35. Signal Hypothesis - Entry into ER • Blobel and Sabatini - explanation for difference between free and membrane-bound ribosomes • All ribosomes have the same structure, distinction dependent upon protein possessing signal sequence

36. Synthesis of secretory proteins 1. N-terminal signal sequence is synthesized 2. Signal bound by SRP, complex docks with SRP receptor on ER membrane 3. Signal sequence binds to translocon, internal channel opens, inserted into translocon 4. Polypeptide elongates, signal sequence cleaved 5. ER chaperones prevent faulty folding, carbohydrates added to specific residues 6. Ribosomes released, recycle 7. C-terminus of protein drawn into ER lumen, translocon gate shuts, protein assumes final conformation