Understanding Membranes: Structures, Proteins, and Functions

1. Chapter 9 Membranes and Cell Surfaces to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

2. Outline • 9.1 Membranes • 9.2 Structure of Membrane Proteins • 9.3 Membrane and Cell-Surface Polysaccharides • 9.4 Glycoproteins • 9.5 Proteoglycans

3. 9.1 Membranes Structures with many cell functions • Barrier to toxic molecules • Help accumulate nutrients • Carry out energy transduction • Facilitate cell motion • Assist in reproduction • Modulate signal transduction • Mediate cell-cell interactions

4. Spontaneously formed lipid structures Hydrophobic interactions all! • Very few lipids exists as monomers • Monolayers arrange lipid tails in the air! • Micelles bury the nonpolar tails in the center of a spherical structure • Micelles reverse in nonpolar solvents

6. Spontaneously formed lipid structures Hydrophobic interactions all! • Lipid bilayers can form in several ways • unilamellar vesicles (liposomes) • multilamellar vesicles (Alex Bangham)

9. The Fluid Mosaic Model S. J. Singer and G. L. Nicolson • The phospholipid bilayer is a fluid matrix • The bilayer is a two-dimensional solvent • Lipids and proteins can undergo rotational and lateral movement • Two classes of proteins: • peripheral proteins (extrinsic proteins) • integral proteins (intrinsic proteins)

11. Motion in the bilayer • Lipid chains can bend, tilt and rotate • Lipids and proteins can migrate ("diffuse") in the bilayer • Frye and Edidin proved this (for proteins), using fluorescent-labelled antibodies • Lipid diffusion has been demonstrated by NMR and EPR (electron paramagnetic resonance) and also by fluorescence measurements

13. Membranes are Asymmetric • Lateral Asymmetry of Proteins: • Proteins can associate and cluster in the plane of the membrane - they are not uniformly distributed in many cases • Lateral Asymmetry of Lipids: • Lipids can cluster in the plane of the membrane - they are not uniformly distributed

15. Membranes are Asymmetric • Transverse asymmetry of proteins • Mark Bretscher showed that N-terminus of glycophorin is extracellular whereas C-terminus is intracellular • Transverse asymmetry of lipids • In most cell membranes, the composition of the outer monolayer is quite different from that of the inner monolayer

17. Flippases A relatively new discovery! • Lipids can be moved from one monolayer to the other by flippase proteins • Some flippases operate passively and do not require an energy source • Other flippases appear to operate actively and require the energy of hydrolysis of ATP • Active flippases can generate membrane asymmetries

19. Membrane Phase Transitions The "melting" of membrane lipids • Below a certain transition temperature, membrane lipids are rigid and tightly packed • Above the transition temperature, lipids are more flexible and mobile • The transition temperature is characteristic of the lipids in the membrane • Only pure lipid systems give sharp, well-defined transition temperatures

22. 9.2 Structure of Membrane Proteins Singer and Nicolson defined two classes • Integral (intrinsic) proteins • Peripheral (extrinsic) proteins • We'll note a new one - lipid-anchored proteins

23. Peripheral Proteins • Peripheral proteins are not strongly bound to the membrane • They can be dissociated with mild detergent treatment or with high salt concentrations

24. Integral Membrane Proteins • Integral proteins are strongly imbedded in the bilayer • They can only be removed from the membrane by denaturing the membrane (organic solvents, or strong detergents) • Often transmembrane but not necessarily • Glycophorin, bacteriorhodopsin are examples

25. Glyophorin A single-transmembrane-segment protein • One transmembrane segment with globular domains on either end • Transmembrane segment is alpha helical and consists of 19 hydrophobic amino acids • Extracellular portion contains oligosaccharides and these constitute the ABO and MN blood group determinants • Mark Bretscher showed that glycophorin was a transmembrane protein

27. Bacteriorhodopsin A 7-transmembrane-segment (7-TMS) protein • Found in purple patches of Halobacterium halobium • Consists of 7 transmembrane helical segments with short loops that interconnent the helices • Note the symmetry of packing of bR (see Figure 9.15) • bR is a light-driven proton pump!

31. Lipid-Anchored Proteins A relative new class of membrane proteins • Four types have been found: • Amide-linked myristoyl anchors • Thioester-linked fatty acyl anchors • Thioether-linked prenyl anchors • Glycosyl phosphatidylinositol anchors

32. Amide-Linked Myristoyl Anchors • Always myristic acid • Always N-terminal • Always a Gly residue that links • Examples: cAMP-dependent protein kinase, pp60src tyrosine kinase, calcineurin B, alpha subunits of G proteins, gag protein of HIV-1

34. Thioester-linked Acyl Anchors • Broader specificity for lipids - myristate, palmitate, stearate, oleate all found • Broader specificity for amino acid links - Cys, Ser, Thr all found • Examples: G-protein-coupled receptors, surface glycoproteins of some viruses, transferrin receptor triggers and signals

35. Thioether-linked Prenyl Anchors • Prenylation refers to linking of "isoprene"-based groups • Always Cys of CAAX (C=Cys, A=Aliphatic, X=any residue) • Isoprene groups include farnesyl (15-carbon, three double bond) and geranylgeranyl (20-carbon, four double bond) groups • Examples: yeast mating factors, p21ras and nuclear lamins

37. Glycosyl Phosphatidylinositol Anchors • GPI anchors are more elaborate than others • Always attached to a C-terminal residue • Ethanolamine link to an oligosaccharide linked in turn to inositol of PI • See Figure 9.20 • Examples: surface antigens, adhesion molecules, cell surface hydrolases

40. Lipid Anchors are Signaling Devices • Recent evidence indicates that lipid anchors are quite transient in nature • Reversible anchoring and de-anchoring can control (modulate) signalling pathways • Similar to phosphorylation/ dephosphorylation, substrate binding/ dissociation, proteolytic cleavagetriggers and signals

41. Bacterial Cell Walls Composed of 1 or 2 bilayers and peptidoglycan shell • Gram-positive: One bilayer and thick peptidoglycan outer shell • Gram-negative: Two bilayers with thin peptidoglycan shell in between • Gram-positive: pentaglycine bridge connects tetrapeptides • Gram-negative: direct amide bond between tetrapeptides

44. More Notes on Cell Walls • Note the gamma-carboxy linkage of isoglutamate in the tetrapeptide • Peptidoglycan is called murein - from Latin "murus", for wall • Gram-negative cells are hairy! Note the lipopolysaccharide "hair" in Figures 9.23 and 9.24

48. Cell Surface Polysaccharides A host of important functions! • Animal cell surfaces contain an incredible diversity of glycoproteins and proteoglycans • These polysaccharide structures regulate cell-cell recognition and interaction • The uniqueness of the "information" in these structures is determined by the enzymes that synthesize these polysaccharides

49. 9.4 Glycoproteins Many structures and functions! • May be N-linked or O-linked • N-linked saccharides are attached via the amide nitrogens of asparagine residues • O-linked saccharides are attached to hydroxyl groups of serine, threonine or hydroxylysine • See structures in Figure 9.26