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.
Outline/Learning Objectives
I. Membrane lipids
A. Membrane functions
B. Isolating membrane lipids
C. Historical models of
membranes
D. Fluid mosaic model
E. 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.
Membrane Functions
Membranes: How would you study them?
Lyse w/ dH2O, centrifuge
Plasma membrane “ghosts”
Extract w/ chloroform-MeOH
Centrifuge
pellet
SDSPAGE:
Proteins separated by size:
smallest travel farthest
supernatant
TLC:
Lipids separated by polarity:
least travels farthest
MB phospholipids
Note: backbone
is glycerol
Note: backbone is serine
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
Fluid Mosaic Model
Singer and Nicholson (1972) Science 175:720
1. Evidence of the phospholipid
composition: TLC of various membranes
Conclusion:
2. Evidence for Lipid Bilayer:
X-ray crystallography of Membranes
10 nm
• X-ray crystallography of
membranes directly reveals
the bilayer structure.
• Polar head groups scatter
electrons more at peaks.
• Distance between peaks is
10 nm.
Data
Interpretation
distance
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
• Membrane asymmetry is generated
– Rotation and lateral diffusion
during synthesis in the ER:
• PC, SM mostly in outer leaflet
• PE, PS, PI mostly in inner leaflet
• Cholesterol: 50% inner, 50% outer
is rapid
– Transverse diffusion or "flipflop" is rare, mediated by
protein translocases.
3. Evidence for Lipid Fluidity:
Fluorescence Recovery After
Photobleaching (FRAP)
Lipids
labeled
4. Evidence for Fluidity:
Differential 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.
Monounsaturated
saturated
• Longer fatty acid chains have a
higher Tm while shorter fatty acids
have a lower Tm (more fluid).
Effects of Chain Length and
Double Bonds on Tm
Less fluid →
More fluid →
Effect of Unsaturated Fatty
Acids on Fluidity
• C=C 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.
MB Fluidity Depends On:
• Temperature
– Higher T, greater fluidity; cells can’t change.
• Unsaturated FAs
– Increase fluidity
• Length of FAs
– Shorter, more fluid
• Cholesterol
– Fluidity “buffer”
Cells can regulate
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 FA’s
immobilized
Makes MB more fluid at lower
temperatures than without
cholesterol, since it disrupts
packing into a crystal.
Summary: Evidence concerning the
Lipid Portion of the Membrane
1.
Estimated and measured surface area
•
2.
Membrane is a bilayer.
Electron microscopy
•
3.
Trilaminar appearance of membranes.
X-ray crystallography
•
4.
Membrane is a bilayer.
Thin-layer chromatography
•
5.
Different membranes contain different phospholipids.
Fluorescence recovery after photobleaching of lipids
•
6.
Membranes are fluid.
Differential scanning calorimetry
•
The phospholipid composition of membranes determines how
fluid they are.
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?
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.
CHO modification of Glycolipids:
The 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:
A antigen
A allele
HH or Hh
Precursor H substance
H Substance
hh
B allele
B antigen
ABO Blood Groups
A - A antigen only
B - B antigen only
AB - Both A and B antigens
O - Neither antigen