Chapter 9 Opener

Lipids

• Signaling molecules, hormones• Vitamins, cofactors• Energy storage (reduced carbon)• Membrane bilayer

• sine qua non for the existence of cells [‘that without which, nothing’]

Figure 9-1

Fatty acids: important components/precursors for membrane lipids

saturated -> unsaturated -> polyunsaturated

‘saturated’ refers to the hydrocarbon tail having no double bonds and therefore being fully saturated with the maximum number of bonds to hydrogen atoms

Text, Table 9-1

Note the dependence of melting temperature on hydrocarbon chain length and degree of unsaturation

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Three fatty acid chains esterifying a glycerol = fat

Text, pg. 249

Figure 9-3

Phospholipids: the most abundant component of the lipid bilayer

Note the double-tail

Text, Figure 9-3

Table 9-2

Common phophoryl substituents in natural phopholipids

Text, Table 9-2

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Variations on the glycerol (amino glycerol) and the fatty acid tail

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Steroids

Text, figures (various)

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Isoprene and isoprenoids

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Note that the isoprene backbones are wrong in the online versions of the textbook material!

The isoprene is a 5 carbon repeating unit

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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.

Figure 9-13

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 9-18

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.

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

lipidic assemblies

Association of proteins with membranes

Prevalence: 20%-25% of all proteins in a given genome

Roles: Transport, signaling, attachment, bilayer remodeling

Categories:1. Transmembrane (or integral membrane)2. Membrane-anchored3. Peripheral

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)

1. Transmembrane Proteins

A short history of structural investigations on transmembrane proteins

The photosynthetic reaction center from Rb. sphaeroides

Two basic kinds of architectures:

1. bundles of -helices crossing the membrane (this case predominates)

2. -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.

1. Transmembrane Proteins

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).

Figure 9-21

Example of a hydropathy plot

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 -helix is ~1.5 Å, so one expects a hydrophobic stretch >20 aa to span the bliayer. [Different for ]

amino acid sequence

Figure 9-21

Example of a hydropathy plot

More typical hydropathy plot: uracil permease, believed to have 10 TM helices(from Matt Talbert)

Figure 9-21

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

Using structural information to understand the TM protein-lipid interface

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Figure 9-21

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

Using structural information to understand the TM protein-lipid interface

Examining the amino acids in the MT region showed that:1. Charged groups were excluded2. The amino acids most exposed to the lipid bilayer (hatched circles) were very nonpolar3. (not shown) The amino acids most exposed to the lipid bilayer tended to be least conserved between related protein sequences

Figure 9-21

The reaction center studies led to general views about TM proteins:

1. 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

2. The lipid-facing parts of TM proteins are extremely hydrophobic; in contrast to soluble proteins, the most hydrophobic parts of TM proteins face outward

3. 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

Using helical wheels to examine amino acid sequences to see if they suggest an alpha helix

with two sides that are very different

from O’Neil and Grisham, Univ. Virginia

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).

Figure 9-21

Different kinds of properties (besides hydrophobicity) can be plotted on a helical wheel

from O’Neil and Grisham, Univ. Virginia

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.

Figure 9-23a

An unusual property of transmembrane proteins still not fully understood: aromatic (esp. Trp)

residues common at the bilayer boundary

Aromatic residues shown in white

2. Membrane-anchored proteins

Types of anchoring:

A. Isoprene linkageB. Fatty acid linkageC. GPI linkage

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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.

A. Isoprene linkage

Figure 9-24

Types: Myristic acid (C14) or palmitic acid (C16)

Attachment:

• Myristoylation – amidation of the fatty acid by the -amino group of N-terminal glycine

• Palmitoylation – thioesterification of the fatty acid by the thiol group of a cysteine sidechain

B. Fatty acid linkage

Figure 9-24

Attachment: amidation of the C-terminus of the protein backbone by the ethanolamine group on the GPI.

C. GPI (glycosyl phosphatidylinositol) - linkage

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