1. Membrane Structure and Dynamics
CH353 February 14, 2008

2. Summary Membrane Lipids Diversity and distribution
Biophysics: phase transitions and diffusion
Structures
Membrane Proteins
Classification
Functions:
Membrane shape
Membrane fusion
Cell adhesion

3. General Properties of Biomembranes
Non-covalent assembly of lipid and protein – fluid mosaic
Lipids spontaneously form a bilayer 5–8 nm thick
Hydrophobic interior with hydrophillic surfaces
Selectively impermeable to polar molecules
Creates a barrier for separating aqueous environments
Movement of lipids and proteins
Rapid diffusion within each monolayer
Slow diffusion from one monolayer to another
Structure and function depends on both lipid and protein
Asymmetry of lipids and proteins in each monolayer
Electrochemical differences across membrane

4. Diversity of Lipid Components
Factors determining fluidity, thickness, shape and activity of biomembrane
Type of Lipid
glycerolipid, sphingolipid, cholesterol
Variations in acyl or ether groups
length, unsaturation
Head Group Alcohol
choline, ethanolamine, inositol, serine, carbohydrate
>1000 different combinations of acyl and head groups per eukaryotic cell
Sprong et al. 2001, Nature Rev. Mol. Biol. 2: 504.

5. Distribution of Lipids in Organelles
Cholesterol in plasma membrane
Cardiolipin in inner mitochondrial
Sphingolipids in lysosomal

6. Distribution of Lipids in Bilayer
Erythrocyte plasma membrane
Inside (anionic groups)
phosphatidylethanolamine
phosphatidylserine
phosphatidylinositols
phosphatidic acid
Outside (neutral groups)
phosphatidylcholine
sphingomyelin
Both
cholesterol

7. Fluidity of Biomembranes
Pure lipids have a phase transition: gel ↔ fluid
paracrystalline ↔ liquid-disordered
Biomembranes having mixtures of lipids exist in liquid-ordered state
Cell changes the composition of its lipid bilayer to maintain that state
Less fluid membranes have longer and more saturated acyl groups
cis double bonds disrupt packing
sterols pack with saturated acyl groups; both ordered and fluid

8. Effect of Cholesterol on Membranes
Cholesterol : Lecithin
0.2
0.3
0.1
0.0 10 20 30 40
Temperature (ºC) so lo ld
Phase Diagram determined by EPR (Electron Paramagnetic Resonance) ld so lo
Meer et al. 2008, Nature Rev. Mol. Biol. 9: 112.

9. Lateral Movement of Lipids
Rapid diffusion of lipids within the monolayer
Motion is restricted by cell structures
Organelles
Cell-cell junctions
Cytoskeletal elements
Fluorescence microscopy of single lipid
Rapid diffusion within a region with jumps to other regions

10. Lateral Movement of Lipids
Fluorescence Recovery After Photobleaching (FRAP) Analysis
Outer leaflet of membrane is labeled with probe
Laser bleaches a spot on labeled lipids
Fluorescence microscopy shows rapid lateral motion of lipids into the bleached spot on membrane

11. Transverse Movement of Lipids
Movement of lipids from one bilayer to another is relatively slow
Biological flipping of lipids is catalyzed with proteins
P4 ATPases
ATP binding cassette (ABC) transporters

12. Movement of Lipids from Monolayer
t1/2 of spontaneous transfer for various lipids
loss of head group alcohol ↑ transbilayer diffusion
loss of fatty acyl group ↑ interbilayer transfer
liquid-ordered domains ↓ both types of transfer
cholesterol shows rapid transbilayer diffusion
Hothuis & Levine 2005, Nature Rev. Mol. Biol. 6: 209.

13. Cross-Sectional Shapes of Lipids
amphipathic molecules with a single hydrocarbon chain form micelles
detergents
fatty acids
lysoglycerophospholipids
some amphipathic molecules with two hydrocarbon chains form bilayers
phosphatidylcholine
some lipids do not form stable bilayers
cholesterol
phosphatidylethanolamine

14. Shapes of Lipids Determine Structures
Lysophosphatidylcholine (lysolecithin) – conical
forms micelles
Phosphatidylcholine (lecithin)
cylindrical
forms lipid bilayer
Phosphatidylethanolamine
inverted conical
forms inverse micelles
(inverted hexagonal phase)
Lipids with non-cylindrical cross sections may have special cellular functions
Sprong et al. 2001, Nature Rev. Mol. Biol. 2: 504.

15. Lipids Determine Membrane Thickness
Phosphatidylcholine bilayer:
3.5 nm thick
Phosphatidylcholine + cholesterol bilayer:
4.0 nm thick
Sphingomyelin + cholesterol bilayer:
4.7 nm thick
Lipid rafts – local regions of thicker membrane with more sphingolipid and cholesterol
Sprong et al. 2001, Nature Rev. Mol. Biol. 2: 504.

17. Types of Membrane Proteins
Integral Proteins
Covalently attached to lipid or embedded in membrane
Require extraction with agents that interfere with hydrophobic interactions, e.g. detergents
Peripheral Proteins
Non-covalent interactions with integral proteins or lipids
Can be removed using mild methods disrupting ionic interactions and H-bonds

18. Types of Integral Membrane Proteins
Type I – one transmembrane helix, N-term outside
Type II – one transmembrane helix, C-term outside
Type III – multiple transmembrane helices on single polypeptide
Type IV – multiple transmembrane helices on separate polypeptides
Type V – proteins covalently bound to lipid
Type VI – proteins with covalently bound lipid and transmembrane helix

19. Glycophorin A type I membrane protein
Amino-terminal domain has polar amino acids and is glycosylated
15 O-linked tetrasaccharides
1 N-linked glycan
Transmembrane domain has hydrophobic amino acids
Carboxy-terminal domain has polar amino acids

20. Bacteriorhodopsin A type III membrane protein
7 transmembrane α-helices
Each helix is composed of hydrophobic amino acids
Loops joining helices have polar amino acids
A light-driven H+ pump
Analogous to rhodopsin
(a G protein-coupled receptor)

21. Prediction of Transmembrane α-Helices
Each amino acid is given a hydropathy index based on free energy of transfer to water
Hydropathy of peptides within an amino acid sequence are calculated and plotted vs residue number
Transmembrane peptides are ones with hydropathy > 0 for 20–25 amino acids

22. Membrane Proteins with β-Barrel Structures
Bacterial β-barrel porins
Transmembrane β-sheets have alternating polar and non-polar amino acids
Cannot use scanning hydropathy method for predicting membrane spanning β-sheets

23. Lipid-Linked Membrane Proteins
Integral proteins can be attached to membrane by covalently bound lipid
Acylated
palmitoyl on Cys or Ser
N-terminal myristoyl
Prenylated
C-terminal farnesyl or geranylgeranyl group
GPI anchor
C-terminal glycosyl phosphatidylinositol

24. Imaging Membrane Ultrastructure Using Electron Microscopy
Freeze-fracture/freeze-etch scanning EM
Specimen rapidly frozen then fractured along plane parallel to lipid bilayers
Sample is then shadowed with platinum and organic material is dissolved
The freeze-etched metal replica is then analyzed by scanning EM
Image of thylakoid membrane

25. Imaging Membrane Ultrastructure Using Atomic Force Microscopy

26. Structure of Membrane Rafts
Microdomains on surface of plasma membrane
Sphingolipid and cholesterol in liquid-ordered state
Surrounding lipid in bilayer in liquid-disordered state
Resistant to solubilization
Segregate proteins based on attached lipid (fatty acyl groups pack better)
Caveolin embedded in some (inward curvature)

27. Structure of Membrane Caveolae
Caveolae “little caves” caused by the binding of numerous caveolins in membrane rafts
Both bilayers are involved in the rafts
May have important functions in membrane trafficking and signal transduction

28. Cell Adhesion Proteins
Cadherins
homophilic interactions
Immunoglobulin-like domains
N-CAM (homophilic)
I-CAM (bind integrin)
Selectins
bind carbohydrate
Integrins
Combinations of α and β
heterophilic interactions with various ligands
2-way signal tranduction (integrin activation)

29. Cell Adhesion Molecules Participate in Leukocyte Activation and Extravasation
Inflammation causes presentation of P-selectin and platelet activating factor
P-selectin interacts transiently with carbohydrate ligands on leukocytes
PAF activates leukocyte causing activation of integrin (inside-out signalling)
Integrins adhere firmly with ICAM-1 and ICAM-2 on endothelial cells

30. Integrin Heterodimeric Complexes
natural combinations of integrin α and β subunits and their ligands

31. Some processes involving membrane fission and fusion

32. Membrane Fusion of Influenza Virus

33. SNARE-Mediated Membrane Fusion

34. SNARE Conformational Cycle
SNARE complexes: 4 parallel α helices each C-term in membrane
Ca2+ required for binding R-SNARE with Q-SNAREs (acceptor)
ATP needed for dissociation of SNARE complexes
Jahn & Scheller 2006, Nature Rev. Mol. Biol. 7: 631

35. Distinct SNARE Combinations for Different Membrane Fusions
Jahn & Scheller 2006, Nature Rev. Mol. Biol. 7: 631