Chapter 9 Opener

1. Lipids • Signaling molecules, hormones • Vitamins, cofactors • Energy storage (reduced carbon) • Membrane bilayer • sine qua non for the existence of cells [‘that without which, nothing’] Chapter 9 Opener

2. Fatty acids: important components/precursors for membrane lipids ‘saturated’ refers to the hydrocarbon tail having no double bonds and therefore being fully saturated with the maximum number of bonds to hydrogen atoms saturated -> unsaturated -> polyunsaturated Figure 9-1

3. Note the dependence of melting temperature on hydrocarbon chain length and degree of unsaturation natural fatty acids are built up from two-carbon fragments, and so have an even number of carbons Text, Table 9-1

4. Three fatty acid chains esterifying a glycerol = fat Text, pg. 249 Page 248

5. Phospholipids: the most abundant component of the lipid bilayer Text, Figure 9-3 Note the double-tail Figure 9-3

6. Common phophoryl substituents in natural phopholipids Text, Table 9-2 Table 9-2

7. Variations on the glycerol (amino glycerol) and the fatty acid tail Page 252

8. Steroids Text, figures (various) Page 254

9. Isoprene and isoprenoids Page 257

10. Note that the isoprene backbones are wrong in the online versions of the textbook material! The isoprene is a 5 carbon repeating unit Page 258

11. Note that the tails from various isoprenes have a recognizable (branching) structure, but otherwise there is considerable variability, particularly with respect to saturation and conjugation. Page 258

12. Why are biological membranes made primarily from (diacyl) phospholipids? (i.e. why two-tails)? Common detergents have a polar head and a single tail, and tend to form micelles. Geometric considerations show that it takes a bulkier tail (e.g. a double tail) to form a bilayer. Text, Figure 9-13 Figure 9-13

13. Lipid bilayers can be well-ordered and semi-crystalline at low temperature, but are fluid at temperatures above a phase transition Biological membrane need to be fluid. Fluidity is controlled by membrane composition. Unsaturated lipid, steroids, and other lipids tend to disrupt the semi-crystalline arrangement of well-packed saturated hydrocarbon tails. Figure 9-18

14. A simple mathematical model for lipid molecule shape and how it relates to various types of lipidic assemblies

15. Association of proteins with membranes • Prevalence: 20%-25% of all proteins in a given genome • Roles: Transport, signaling, attachment, bilayer remodeling • Categories: • Transmembrane (or integral membrane) • Membrane-anchored • Peripheral

16. 1. Transmembrane Proteins • A short history of structural investigations on transmembrane proteins • generally hard to study • low solubility and stability presents a problem • very difficult to crystallize • late 70’s: Henderson and Unwin, et al. grew 2D crystals of bacteriorhodopsin (7 transmembrane helices) and obtained resolution by EM high enough to see the TM helices • early 80’s: good 3D crystals of photosynthetic reaction centers were grown first by a German team (Huber, Michel, Deisenhofer) and then from a different organism by a group at UCSD, who did their structural work with a group at UCLA • The German team published the first atomic resolution structures of the reaction center in 1984; the UCSD/UCLA group in 1986. (Nobel prize in 1988 to the German team) • Now ~100 transmembrane protein structures known • Nobel prize for the first channel structure in 2003 (Agre & MacKinnon)

17. 1. Transmembrane Proteins A short history of structural investigations on transmembrane proteins The photosynthetic reaction center from Rb. sphaeroides

18. 1. Transmembrane Proteins • Two basic kinds of architectures: • bundles of a-helices crossing the membrane (this case predominates) • b-sheet wrapped into a barrel (so there aren’t any unsatisfied edge strands) • The key constraint is that the hydrogen bonding requirements of the protein backbone need to be satisfied internally; bulk water is not available to hydrogen bond to the part of the backbone not involved in regular secondary structure (i.e. in loops). This is in contrast to ‘soluble’ proteins.

19. Transmembrane Proteins – prediction from sequence Proteins (or protein domains) that exist in the bilayer have distinctive properties (compared to soluble proteins) that can often be detected from an examination of amino acid sequence. It was recognized before TM structures were known that segments of a protein that passed through the membrane were highly hydrophobic (i.e. non-polar). This is often analyzed in a ‘hydropathy plot’ (Russ Doolittle, UCSD).

20. Example of a hydropathy plot amino acid sequence Note that the analysis is based on the amino acid sequence. The quantity evaluated is usually the average hydrophobicity of a segment of the sequence (e.g. a 20 residue sliding window). This smooths out the noise and gives more interpretable plots). Also note, the bilayer thickness is ~30-40 Å. The rise per amino acid an a-helix is ~1.5 Å, so one expects a hydrophobic stretch >20 aa to span the bliayer. [Different for b] Figure 9-21

21. Example of a hydropathy plot More typical hydropathy plot: uracil permease, believed to have 10 TM helices (from Matt Talbert) Figure 9-21

22. Using structural information to understand the TM protein-lipid interface The first analysis of a TM protein surface and its implications for being in the lipid bilayer. Yeates, Komiya, Rees, Allen, and Feher (1987). Structure of the reaction center from Rhodobacter sphaeroides R-26: membrane-protein interactions.Proc Natl Acad Sci 84, 6438–6442. Figure 9-21

23. Using structural information to understand the TM protein-lipid interface 90° colored by atom type (carbon = green; nitrogen = blue; oxygen = red) Figure 9-21

24. Using structural information to understand the TM protein-lipid interface The surface of the reaction center showed a region about 40Å thick where the surface exposed atoms were very non-polar (i.e. carbon atoms). [The calculation was done in 5 Å slabs to get a smooth signal] The first analysis of a TM protein surface and its implications for being in the lipid bilayer. Yeates, Komiya, Rees, Allen, and Feher (1987). Structure of the reaction center from Rhodobacter sphaeroides R-26: membrane-protein interactions.Proc Natl Acad Sci 84, 6438–6442. Figure 9-21

25. Using structural information to understand the TM protein-lipid interface Examining the amino acids in the MT region showed that: 1. Charged groups were excluded 2. The amino acids most exposed to the lipid bilayer (hatched circles) were very nonpolar 3. (not shown) The amino acids most exposed to the lipid bilayer tended to be least conserved between related protein sequences Figure 9-21

26. The reaction center studies led to general views about TM proteins: • The interior regions (i.e. those not in contact with lipid) of TM proteins are much like regular soluble proteins in terms of composition: fairly non-polar • The lipid-facing parts of TM proteins are extremely hydrophobic; in contrast to soluble proteins, the most hydrophobic parts of TM proteins face outward • The lipid-facing parts of TM proteins are most variable in sequence. This parallels the soluble protein case in that regions interior to the protein are least free to vary; they are more constrained. Figure 9-21

27. Using helical wheels to examine amino acid sequences to see if they suggest an alpha helix with two sides that are very different In this example, the two sides have different hydrophobicities. This can happen for a helix at the surface of a soluble protein or for a transmembrane helix; for the TM case there would be a very hydrophobic side facing the lipid, while for the soluble case there will be a polar side facing the solvent (water). from O’Neil and Grisham, Univ. Virginia Figure 9-21

28. Different kinds of properties (besides hydrophobicity) can be plotted on a helical wheel For example, if one plots the degree of sequence variation when compared to similar (homologous) sequences from other organisms, one may see a difference between sequence variability on two sides of a helix. The side that is most conserved is implied to face in to the protein interior, while the variable side is expected to face outward (to the solvent for a soluble protein or to the lipid for a TM protein). These problems can be treated mathematically. from O’Neil and Grisham, Univ. Virginia Figure 9-21

29. An unusual property of transmembrane proteins still not fully understood: aromatic (esp. Trp) residues common at the bilayer boundary Aromatic residues shown in white Figure 9-23a

30. Membrane-anchored proteins • Types of anchoring: • Isoprene linkage • Fatty acid linkage • GPI linkage

31. A. Isoprene linkage Recognition sequence: C-terminal C-X-X-Y (X aliphatic, Y affects which type of lipid gets attached). Then, X-X-Y gets cleaved, followed by methyl esterification of new C-terminus. Page 268

32. B. Fatty acid linkage • Types: Myristic acid (C14) or palmitic acid (C16) • Attachment: • Myristoylation – amidation of the fatty acid by the a-amino group of N-terminal glycine • Palmitoylation – thioesterification of the fatty acid by the thiol group of a cysteine sidechain Figure 9-24

33. C. GPI (glycosyl phosphatidylinositol) - linkage Attachment: amidation of the C-terminus of the protein backbone by the ethanolamine group on the GPI. Figure 9-24

34. 3. Peripheral membrane proteins Bound to membrane by interactions at the bilayer surface Usually can be dissociated from the membrane relatively easily (e.g. high salt), and are typically soluble afterwards