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Membrane Structure and Dynamics. CH353 February 14, 2008. Summary. Membrane Lipids Diversity and distribution Biophysics: phase transitions and diffusion Structures Membrane Proteins Classification Structures Functions: Membrane shape Membrane fusion Cell adhesion.

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Membrane structure and dynamics

Membrane Structure and Dynamics

CH353 February 14, 2008


Summary
Summary

Membrane Lipids

  • Diversity and distribution

  • Biophysics: phase transitions and diffusion

  • Structures

    Membrane Proteins

  • Classification

  • Structures

  • Functions:

    • Membrane shape

    • Membrane fusion

    • Cell adhesion


General properties of biomembranes
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


Diversity of lipid components
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.


Distribution of lipids in organelles
Distribution of Lipids in Organelles

  • Cholesterol in plasma membrane

  • Cardiolipin in inner mitochondrial

  • Sphingolipids in lysosomal


Distribution of lipids in bilayer
Distribution of Lipids in Bilayer

Erythrocyte plasma membrane

  • Inside (anionic groups)

    • phosphatidylethanolamine

    • phosphatidylserine

    • phosphatidylinositols

    • phosphatidic acid

  • Outside (neutral groups)

    • phosphatidylcholine

    • sphingomyelin

  • Both

    • cholesterol


Fluidity of biomembranes
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


Effect of cholesterol on membranes

Phase Diagram determined by EPR (Electron Paramagnetic Resonance)

ld

40

ld

so

30

lo

Temperature (ºC)

20

lo

so

10

0

0.0

0.1

0.2

0.3

Cholesterol : Lecithin

Effect of Cholesterol on Membranes

Meer et al. 2008, Nature Rev. Mol. Biol. 9: 112.


Lateral movement of lipids
Lateral Movement of Lipids Resonance)

  • 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


Lateral movement of lipids1
Lateral Movement of Lipids Resonance)

  • 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


Transverse movement of lipids
Transverse Movement of Lipids Resonance)

  • 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


Movement of lipids from monolayer
Movement of Lipids from Monolayer Resonance)

  • 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.


Cross sectional shapes of lipids
Cross-Sectional Shapes of Lipids Resonance)

  • 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


Shapes of lipids determine structures
Shapes of Lipids Determine Structures Resonance)

  • Lysophosphatidylcholine (lysolecithin) – conical

    • forms micelles

  • Phosphatidylcholine (lecithin)

    • cylindrical

    • forms lipid bilayer

  • Phosphatidylethanolamine

    • inverted conical

    • forms inverse micelles

      (inverted hexagonal phase)

  • Lipids with non-cylindricalcross sections may have special cellular functions

Sprong et al. 2001, Nature Rev. Mol. Biol. 2: 504.


Lipids determine membrane thickness
Lipids Determine Membrane Thickness Resonance)

  • 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.


Types of membrane proteins
Types of Membrane Proteins Resonance)

  • 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


Types of integral membrane proteins
Types of Integral Membrane Proteins Resonance)

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


Glycophorin
Glycophorin Resonance)

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


Bacteriorhodopsin
Bacteriorhodopsin Resonance)

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)


Prediction of transmembrane helices
Prediction of Transmembrane Resonance)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


Membrane proteins with barrel structures
Membrane Proteins with Resonance)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


Lipid linked membrane proteins
Lipid-Linked Membrane Proteins Resonance)

  • 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


Imaging membrane ultrastructure using electron microscopy
Imaging Membrane Ultrastructure Using Electron Microscopy Resonance)

  • 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



Structure of membrane rafts
Structure of Membrane Rafts Microscopy

  • 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)


Structure of membrane caveolae
Structure of Membrane Caveolae Microscopy

  • 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


Cell adhesion proteins
Cell Adhesion Proteins Microscopy

  • 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)


Cell adhesion molecules participate in leukocyte activation and extravasation
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


Integrin heterodimeric complexes
Integrin Heterodimeric Complexes and Extravasation

natural combinations of integrin α and β subunits and their ligands





Snare conformational cycle
SNARE Conformational Cycle and Extravasation

  • 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


Distinct snare combinations for different membrane fusions
Distinct SNARE Combinations for Different Membrane Fusions and Extravasation

Jahn & Scheller 2006, Nature Rev. Mol. Biol. 7: 631


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