Announcements, Feb. 9

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Announcements, Feb. 9

• Reading for today 154-171 on membrane lipids.
• Reading for Monday 172-186 on membrane proteins.
• Reading for Wednesday 191-207 on membrane
  transport.
• Reading for Friday 207-216 on energetics of
  membrane transport.

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Outline/Learning Objectives

• I. Membrane lipids
• Membrane functions
• Isolating membrane lipids
• Historical models of membranes
• Fluid mosaic model
• Evidence concerning lipid part of membrane

• After reading the text, attending lecture, and
  reviewing lecture notes, you should be able to
• List various functions of membranes.
• Explain how thin-layer chromatography (TLC) can
  be used to fractionate lipids.
• Compare historical models of membrane structure.
• Describe experimental evidence for membrane lipid
  composition, structure and fluidity.

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Membrane Functions
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Membranes How would you study them?
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MB phospholipids
Note backbone is glycerol
Note back- bone is serine
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Historical models of membrane structure

• Gorter and Grendel (1925)
• Estimated red cell surface area and extracted
  lipid from "ghosts."
• Predicted that area of RBC was 100 ?m2, found
  that area covered by lipid was 200 ?m2 ,
  indicating a bilayer
• Davson and Danielli Model (1935)
• How does differential permeability come about?
• Proposed lipid bilayer protein lamellae on each
  side (sandwich), pores allowed substances in or
  out.
• Robertson (1960)
• Viewed membranes with EM, seemed to agree with
  Davson and Danielli model
• Suggested that all membranes of the same
  composition (unit membrane).
• But unit MB model did not account for chemical
  differences in membranes

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Fluid Mosaic Model Singer and Nicholson (1972)
Science 175720
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1. Evidence of the phospholipid composition TLC
of various membranes
Conclusion
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2. Evidence for Lipid BilayerX-ray
crystallography of Membranes

• X-ray crystallography of membranes directly
  reveals the bilayer structure.
• Polar head groups scatter electrons more at
  peaks.
• Distance between peaks is 10 nm.

10 nm
electron density
Data
distance
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Asymmetry and Movement of PLs

• Functional significance
• Contributes to net negative charge on inside
• PI is available for signaling function on inside.
• Glycolipids in outer leaflet, so CHO out.
• Inequality is maintained by movement properties
  of phospholipids within the membrane
• Rotation and lateral diffusion is rapid
• Transverse diffusion or "flip-flop" is rare,
  mediated by protein translocases.

• Membrane asymmetry is generated during synthesis
  in the ER
• PC, SM mostly in outer leaflet
• PE, PS, PI mostly in inner leaflet
• Cholesterol 50 inner, 50 outer

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3. Evidence for Lipid FluidityFluorescence
Recovery After Photobleaching (FRAP)
Lipids labeled
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4. Evidence for FluidityDifferential Scanning
Calorimetry

• Measures uptake of heat during phase transitions
  of lipids.
• Below the transition temperature (Tm) lipids are
  solid, above Tm lipids are fluid.
• Saturated fatty acids have a higher Tm while
  unsaturated fatty acids have a lower Tm (more
  fluid). Why?
• Double bonds make kinks in the tails, which
  disrupt the crystal structure.
• Longer fatty acid chains have a higher Tm while
  shorter fatty acids have a lower Tm (more fluid).

Monoun- saturated
saturated
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Effects of Chain Length and Double Bonds on Tm
More fluid ?
Less fluid ?
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Effect of Unsaturated Fatty Acids on Fluidity

• CC in FA creates kinks in chain, so they pack
  together less well.
• Less able to form crystalline solid, therefore
  stays liquid.
• Organisms in cold environments increase the of
  unsaturated FAs in their membranes.

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MB Fluidity Depends On

• Temperature
• Higher T, greater fluidity cells cant change.
• Unsaturated FAs
• Increase fluidity
• Length of FAs
• Shorter, more fluid
• Cholesterol
• Fluidity buffer

Cells can regulate
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Effect of Cholesterol on Fluidity

• Animal cells contain up to 50 cholesterol in
  their membranes.
• OH of cholesterol hydrogen bonds with O of ester
  bonded fatty acid, while hydrocarbon rings
  interact with hydrophobic hydrocarbon chains of
  fatty acids

Acts as a fluidity buffer Makes MB less fluid at
higher temperatures than without cholesterol,
since FAs immobilized Makes MB more fluid at
lower temperatures than without cholesterol,
since it disrupts packing into a crystal.
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Summary Evidence concerning the Lipid Portion of
the Membrane

• Estimated and measured surface area
• Membrane is a bilayer.
• Electron microscopy
• Trilaminar appearance of membranes.
• X-ray crystallography
• Membrane is a bilayer.
• Thin-layer chromatography
• Different membranes contain different
  phospholipids.
• Fluorescence recovery after photobleaching of
  lipids
• Membranes are fluid.
• Differential scanning calorimetry
• The phospholipid composition of membranes
  determines how fluid they are.

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A recent twist on the Fluid Mosaic Model Lipid
rafts
Or Outside cell

• Small, specialized areas in membrane where some
  lipids (primarily sphingolipids and cholesterol)
  and proteins are concentrated.
• Two monolayers move together thicker, less fluid
  than normal membrane
• Function signaling and/or transport of membrane
  proteins?

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Visualization of Lipid Rafts
Atomic force microscopy reveals sphingomyelin
rafts (orange) protruding from a PC background
(black) in a mica-supported lipid bilayer.
Placental alkaline phosphatase (yellow peaks), a
GPI-anchored protein, is shown to be almost
exclusively raft-associated. For details see the
article by Saslowsky et al. J. Biol. Chem. 277,
Cover of 30, 2002.
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CHO modification of GlycolipidsThe ABO blood
groups

• Glycolipids partition into lipid rafts on
  non-cytosolic side
• Sugars added in lumen of Golgi, e.g. AB
  antigens.
• Recall the genetics

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ABO Blood Groups
A - A antigen only B - B antigen only AB -
Both A and B antigens O - Neither antigen