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 lipidsA. Membrane functionsB. Isolating membrane lipidsC. Historical models of

membranesD. Fluid mosaic modelE. 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?

Plasma membrane “ghosts”Lyse w/ dH2O, centrifuge

supernatant

Lipids separated by polarity:least travels farthest

TLC:

pellet

Extract w/ chloroform-MeOHCentrifuge

SDS-PAGE:

Proteins separated by size:smallest travel farthest

MB phospholipids

Note: back-bone is serine

Note: backboneis glycerol

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

• X-ray crystallography of membranes directly reveals the bilayer structure.

• Polar head groups scatter electrons more at peaks.

• Distance between peaks is 10 nm.

distance

elec

tron

de n

s it y

10 nm

Data

Interpretation

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

3. Evidence for Lipid Fluidity:Fluorescence Recovery After

Photobleaching (FRAP)

Lipidslabeled

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.

• Longer fatty acid chains have a higher Tm while shorter fatty acids have a lower Tm (more fluid).saturated

Monoun-saturated

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 immobilizedMakes 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• Membrane is a bilayer.

2. Electron microscopy• Trilaminar appearance of membranes.

3. X-ray crystallography• Membrane is a bilayer.

4. Thin-layer chromatography• Different membranes contain different phospholipids.

5. Fluorescence recovery after photobleaching of lipids• Membranes are fluid.

6. Differential scanning calorimetry• The phospholipid composition of membranes determines how

fluid they are.

A recent twist on the Fluid Mosaic Model: Lipid rafts

• 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?

OrOutside cell

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

Precursor H substance H Substance

A antigen

B antigen

A allele

B allele

HH or Hh

hh

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

ABO Blood Groups

A - A antigen only

B - B antigen only

AB - Both A and B antigens

O - Neither antigen