BMB 170 Lecture 14Lipids and Membranes, Nov 9th

Electron tomographic reconstructionsThe Boulder Laboratory for 3D Electron Microscopy of Cells

http://bio3d.colorado.edu

Part of a yeast cell

C. jejuni PglB

Knockout

Treponema tomogram (Jensen)

“Hydrophobic effect” and lipid bilayers

“Ben Franklin Stilled the Waves” C. Tanford, Oxford (2004)

• Oil on water experiments - Benjamin Franklin 1774, Lord Rayleigh 1890, Agnes Pockels 1892, Irving Langmuir 1917

• Cells– Have a barrier that contains cell contents

• Impermeable to large molecules and small polar molecules

• Permeable to Water• Some molecules must be able to cross the membrane!

– Contain two types of lipids (France early 1800s)• Constant – phospholipids• Variable – fats and oils (storage)

“Hydrophobic effect” and lipid bilayers

“Ben Franklin Stilled the Waves” C. Tanford, Oxford (2004)

• Charles Overton (1899)– Cells completely permeable to many neutral molecules (cell type

independent – anesthetics on tadpoles)– Made the connection that increasing hydrophilicity decreased

permeability– Compared to oil permeability (membranes are similar to oils!)

• Gorter and Grendel (1924)– Gorter was a famous pediatrician – “We propose to demonstrate in this paper that the chromocytes of

different animals are covered by a layer of lipoids just two molecules thick.” J Exp Med

• Measured red blood cell surface area by microscopy• Isolated membranes and measured lipid surface area • Argued that the membrane would be similar to fatty acid crystals (Bragg

1924)

“Hydrophobic effect” and lipid bilayers

“Ben Franklin Stilled the Waves” C. Tanford, Oxford (2004)

• Danielli and Davson (1935)

• Robertson (1959)

The Fluid Mosaic Model

Singer & Nicolson Science (1972) 175:720-31

• Phospholipids assemble into a bilayer through the “hydrophobic effect”– Interior is apolar (largely impermeable to polar molecules - water, ions…)

• Proteins required for transport of polar molecules across membranes– There are peripheral and integral membrane proteins– They have apolar surfaces

Lipid forms

Fig 4.1

Components of sphingomyelin

Fig. 4.6

Phosphocholine(w/o a ceramide)

Amino-alcohol sphingosine

Fatty acid (R-aliphatic chain)

#1#2

headgroups:

from R.B. Gennis “Biomembranes”

Types of Lipids

R

Chain lengths vary from 12 to 22 with up to 4 double bonds

Chiral center

CH2 CH2

O

HC

O

O P OCH2CH2NH3+

O-

O

CO CO

R1 R2

glycerol phosphoester headgroup

fatty acid

Lipid classification

Category Abbr

Fatty acyls FA

Glycerolipids GL

Glycerophospholipids GP

Sphingolipids SP

Sterol lipids ST

Prenol lipids PR

Saccharolipids SL

Polyketides PK

(Z cis bond, E trans bond)

Fahy et al (2005) J Lipid Research 46:839-62

Fig. 4.3

Common acyl chains

Fig. 4.5

DP-dipalmitoylPO-palmitoyloleoylPL-palmitollinoleoylPA-palmitoyarachidonylPD-palmitoyldocosahexaenoyl

• Phospholipid bilayer

• Thermophilic archaea– Increased hydrophobicity– Ethers (not esters) – more stable

• Hyperthermophilic archaea

Archaeal lipidsO

O

PO

OO

OOO

P OO

OOO

P OO

PO

OOO

P OO

PO

Lipid composition

L β L α

Nagle & Tristram-Nagle COSB (2000)10:474-80

Lipid phasesTempGel Phase:

tightly packed, ordered chains & thicker

Liquid crystalline: most of biological membranes, disordered tails (Gennis, p40)

Liquid crystalline DPPC area

Helix ~80Å2

Liquid crystalline & gel phasesPhase Dimension Name Order

Lamellar One Lα Disordered, fluid

Ripple gel Two; oblique or centered Pβ’ Rippled

Gel One Lβ All trans chains

Normal hexagonal Two HI Disordered, fluid oil-in-water

Reversed hexagonal Two HII Disordered, fluid water-in-oil

Cubic Three I Disordered, fluid

Normal Cubic Three II Disordered, fluid

Reversed Cubic Three III Disordered, fluid

Table 4.2Fig. 4.8

Phase diagram• Thermotropic – phase

changes based on temperature

• Lyotropic – can form liquid crystal phasesPhase NameLamellar LαRipple gel Pβ’

Gel LβNormal hexagonal HI

Reversed hexagonal HII

Cubic I

Normal Cubic IIReversed Cubic III Fig. 4.7

Lipid shape affects curvature• Packing parameter: P=v/al• l – length of hydrophobic

chain• a – optimal cross-sectional

area of the head group• When P≅1 (cylinders) you

get bilayers• P>1 curve towards water• P<1 curve away

Fig. 4.11

Lipid structures

a can be dependent on temperature, concentration, etc…

Fig 4.12

More structures

Fig. 4.14 & 4.15

Bicontinuous cubic phasesShape affects radius

Not empty space

Membrane fluidity• Pulling on the

membrane with laser tweezers

• Movie by Steven Block (Stanford)

Heller, Schaeffer & Schulten J Phys Chem (1993) 97:8343-60

20 Å

40 Å

Molecular dynamics simulation of 200 lipids in a bilayer

Bilayer distribution

Lewis & Engelman JMB (1983)166:211

20Å

32Å

capsid

bilayer

DNA

Stuart lab, Cockburn et al Nature (2004) 432:122

• Structure of bacteriophage PRD1– showed boundaries of membrane and DNA

• Dimensions of membrane– most hydrophobic region ~ 20Å– peak to peak distance ~ 32Å; expect ~36Å (10% thinning)– Phospholipid asymmetry

• zwitterionic PE in inner leaflet (DNA)• anionic PG and Cardiolipin in outer leaflet.

4Å Crystal structure of enveloped virus

Bacteria grow in a window

E. coli example

Asymmetric layers

Devaux Annu Rev Biophys Biomol Struct (1992) 21:417-39

Asymmetry can drive structure

• Giant vesicles of phosphatidylcholine

• 1% phosphatidylglycerol and raise pH (4.5-9.5)

• 0.1% redistribution

Farge & Devaux Biophys J (1992) 61, 347-57

Lipids crossing membrane▪ Flipping is intrinsically slow

• Hours to weeks across artificial membranes• Tens of seconds or less across biogenic

membranes▪ Flippases▪ Glycosylated lipids may never cross

Sanyal & Menon (2009) Chem Bio 4:895

Flipping mechanisms

Pore model Slip-pop model for single TMs

Isoprenoid based flippases?

Review: Sanyal & Menon (2009) Chem Bio 4:895MsbA structure: Ward..Chang (2007) PNAS 104:19005

Membrane remodeling is dynamic!• Interplay between lipids and proteins

• Can be temporary or permanent• Can be local or wider region• Functions of membrane

curvature‣ Movement‣ Division‣ Vesicle trafficking/ viral budding/

tubule carrier formation

McMahon & Gallop Nature Review (2005) 438: 590

Lipids are dynamic systems

• Mixed saturated (blue), unsaturated (black) and cholesterol

• Phase separation of saturated lipids and cholesterol from the unsaturated lipids

Tristan Ursell, Phillips lab (now a Prof at U of Oregon)

Lipids partition in model systems

Hancock Nature Rev Mol Cell Biol (2006) 7:456

Rinia & de Kruijff FEBS Let (2001) 504:194

Cholesterol-induced lateral phase separation and phases

Figure 4.28 & 4.29

▪ Saturated phospholipid, cholesterol and water▪ ld – liquid-disordered phase

▪ lo- liquid-ordered phase

▪ so- gel phase

▪ Tm – temperature at the phase transition from gel to lamellar

Lipid rafts▪ Compartmentalization of cellular membranes into lateral subdivision▪ Small (10–200 nm), heterogeneous, highly dynamic, sterol- and

sphingolipid-enriched domains that compartmentalize cellular processes▪ Enriched with cholesterol, sphingolipids, and proteins▪ Drive signal transduction, lipid trafficking, cytoskeletal organization, and

viral budding and entry▪ The true nature of membrane organization in living cells remains unknown

Levental & Veatch J Mol Biol (2016)Pike J Lipid Res (2003) 44:655

Munro Cell (2003) 115:377

Lipid rafts are in liquid ordered (lo) phase:• The acyl chain of

sphingolipids is typically saturated

• H-bonding interaction stabilizes sphingolipid molecules

• Cholesterol orders the acyl chains

• Tight packing, less fluidity, phase separation

• Insolubility in nonionic detergents

▪ Intrinsic factors: a, b, e ▪ Extrinsic factors: c, d

▪ Positive curvature: early stages of vesicle budding

▪ Negative curvature: viruses budding out of the cell

McMahon & Gallop Nature Review (2005) 438: 590

Sensing membrane curvatureBar Domains Reticulons

Amphiphysin BAR structure (1uru)

Class of proteins that drive membrane asymmetryrequired for the ER tubulation

McMahon lab: Peter et al Science (2004) 303:495 Rapoport lab: Voeltz et al Cell (2006) 124:573

Lateral pressure profile

Figure 4.17

At equilibrium, all the different forces exerted on the lipid bilayer have to cancel (total pressure = zero)

Effect on a protein

Cantor (1997) J Phys Chem B 101:1723-5

Effect on a protein

Perozo & Rees (2003) Curr Op Struct Biol 13:432

▪ A prokaryotic mechanosensitive channel (MscL)

Mattress model and protein sorting

▪ Cholesterol induces protein sorting

Mouritsen & Bloom Biophys J (1984) 46:141 Lundbaek et al Biophys J (2003) 84:2080

▪ Protein/membrane hydrophobic size inequality can affect protein aggregation

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Membrane diffusion

• First calculated by Saffman & Delbrück (1975) PNAS– Continuum hydrodynamic model– Diffusion weakly correlated to radius

• Confirmed by GUV measurements– Cell length = 2μm– Surface area 6 μm2

– Rafts/Oligomerization/cytoskeleton μ – viscosity of membraneμ' – viscosity of solventR – radius of moleculeh – bilayer thickness

Ramadurai..Poolman (2009) JACS 131:12650

Fatty acid synthase (type I)Thermomyces lanuginosus

Jenni..Ban (2007) Science 316:254Figure 5.27-5.29

▪ ⍺6β6 dodecamer▪ 2.6 MDa

Domain organization

Fig. 5.29

Self contained reaction chamber

Figure 5.30 & 5.31 Jenni..Ban (2007) Science 316:254