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A.The Phospholipid Bilayer. 1.Lipid Structure2.Experimental systems3.Properties of Lipid Bilayers. A.1.Lipid Structure. Glycerol-based PhospholipidsGlycerol MoleculeTwo Fatty Acid ChainsPolar Head Group, attached via phosphateThe fatty acid chains may be saturated or unsaturatedDouble
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1. Membrane Structure and Transport Processes
A. The Phospholipid Bilayer
B. Membrane Proteins and Carbohydrates
C. Diffusion and Active Transport
D. Electrical Properties of Membranes
2. A. The Phospholipid Bilayer
1. Lipid Structure
2. Experimental systems
3. Properties of Lipid Bilayers
3. A.1. Lipid Structure Glycerol-based Phospholipids
Glycerol Molecule
Two Fatty Acid Chains
Polar Head Group, attached via phosphate
The fatty acid chains may be saturated or unsaturated
Double bonds in unsaturated fatty acids may be cis or trans
4. A.1. Lipid Structure
5. A.1. Lipid Structure Sphingosine-based Lipids
Sphingomyelin
Galactocerebroside
Gangliosides
Cholesterol
6. A. 2. Experimental Systems Liposomes
Artificial vesicles made by mixingpure phospholipidsin water
7. A. 2. Experimental Systems Black Membranes
Artificial lipid bilayer formed between two chambers containing aqueous solutions
Erythrocyte Membranes
8. A. 2. Experimental Systems Erythrocyte Membranes
Separate erythrocytes from blood plasma by centrifugation
Suspend the erythrocytes in a hypotonic buffer
The erythrocytes swell and burst to produce erythrocyte ghosts
By adjusting buffer conditions, sealed ghosts (either normal or inverted) or leaky ghosts can be formed.
9. A. 3. Properties of Lipid Bilayers Overall Structure
Fluidity
Lateral Diffusion of phospholipids: ~10-8 cm2/sec
Rotation
Flexion
Transverse Diffusion of phospholipids: Almost nonexistent. Movement of PL from one leaflet to the other requires enzymes called phospholipid translocators (flippases and scramblases)
10. A. 3. Properties of Lipid Bilayers Measurements of Fluidity
Differential Scanning Calorimetry (DSC)
Determination of the melting point at which a bilayer undergoes phase transition
Electron Spin Resonance (ESR) spectroscopy
Attaching an ESR label such as a nitroxyl group to the fatty acid chain; can detect motility and other interactions within the lipid core of the bilayer
Tagged lipid / real time video microscopy
Attach gold particle or fluorescent group to individual lipid molecules and track movement via digitally enhanced imaging
Laser photobleaching experiments
11. A. 3. Properties of Lipid Bilayers Factors Affecting Fluidity
Length of the fatty acid side chains
Presence of double bonds in the fatty acid chains (degree of saturation)
Size of the polar head groups
Presence of cholesterol
http://www.nyu.edu/pages/mathmol/library/lipids/
12. A. 3. Properties . . .
13. A. 3. Properties . . .
14. A. 3. Properties . . .
15. A. 3. Properties of Lipid Bilayers Compositional Symmetry
Refers to the percentages of each phospholipid in the inner and outer leaflets of the bilayer
In artificial liposomes: Leaflets have exactly the same composition
In membranes: The compositions are different (Asymmetric)
Determined by vectorial labeling experiments
17. A. 3. Properties of Lipid Bilayers Erythrocyte Membrane
(numbers are % of total lipid)
Outside Inside
Sphingomyelin 20% 5%P. choline 25% 5%P. ethanolamine 5% 25%P. serine 0% 5%
18. A. 3. Properties of Lipid Bilayers Lipid rafts
Areas in a bilayer where specific lipids are more concentrated
In a liposome consisting of 1:1:1 phosphatidylcholine:sphingomyelin:cholesterol, the sphingomyelin and cholesterol will form patches that may be similar to lipid rafts
There is evidence that some integral membrane proteins may require specific lipid molecules for activity
19. A. 3. Properties of Lipid Bilayers Role of compositional asymmetry
Charge differences between outer & inner surfaces see difference in distribution of PC and PS in the erythrocyte membrane
Binding and activation of cell signaling proteins
Protein kinase C binds to negative cytosolic face
Phosphatidylinositol can be modified to create specific binding sites for signaling proteins, either by adding phosphates to the inositol or by cleaving PI
20. A. 3. Properties of Lipid Bilayers Role of compositional asymmetry
Compositional asymmetry is used in mammals to detect cells that have undergone apoptosis (programmed cell death)
When the cell dies, the phospholipid translocator that moves PS to the cytosolic leaflet is inactivated
A scramblase that moves phospholipids across the membrane nonspecifically in both directions is activated
PS rapidly moves from the cytosolic leaflet to become equally distributed between the cytosolic leaflet and the exterior leaflet
21. B. Membrane Proteins and Carbohydrates
1. Membrane Proteins
2. Mobility of Membrane Proteins
3. Membrane Carbohydrates
22. B. 1. Membrane Proteins Peripheral, Integral, and Lipid-Anchored Proteins
Solubilization experiments
Peripheral protein: Can be removed by high ionic strength wash
Integral protein: Requires detergent treatment to be solubilized
Lipid-Anchored Protein: Solubilized by detergent or by enzymatic lysis from lipid anchor
23. B. 1. Membrane Proteins It was predicted that transmembrane domains would be rich in hydrophobic amino acids
This was confirmed by amino acid sequencing, then later by determination of integral 3-D structures
The two major transmembrane configurations are a-helical segments and ß-pleated sheets that form beta barrel structures
24. B. 1. Membrane Proteins Examples of Membrane Proteins
Erythrocyte Membrane Proteinshttp://www.unipv.it/bioscipv/sds-page.htm
Integral Proteins
Band 3
Glycophorin A
Peripheral Proteins Membrane Skeleton
Spectrin
Ankyrin
Band 4.1
Actin
25. B. 1. Membrane Proteins Bacteriorhodopsin
The first integral membrane protein in which the 3-D structure was determined
This is a light-driven hydrogen ion pump found in the plasma membrane of the archaen Halobacterium salinarum
Located in specialized regions called purple patches where it is found in crystalline-like arrays
This unique structure allowed its 3-D structure to be determined by electron diffraction analysis
Its transmembrane domain consists of seven a-helical segments
26. B. 1. Membrane Proteins Bacterial porins
Found in the outer membrane of gram-negative bacteria
Transmembrane domain is a beta barrel structure consisting of a ring of ß-pleated sheets that form a large channel
27. B. 2. Mobility of Membrane Proteins Membrane proteins often exhibit lateral mobility, but not transverse mobility (flip-flop)
Demonstrations of lateral mobility
Patching-and-capping of immunoglobulins on B-lymphocytes http://www3.niaid.nih.gov/labs/aboutlabs/lig/lymphocyteActivationSection/
Cell fusion experiments
Photobleaching experiments (FRAP)
Single particle tracking experimentshttp://www.censsis.neu.edu/public_docs/13b-07.pdf
28. B. 2. Mobility of Membrane Proteins Lateral mobility of integral proteins may be limited by interactions with peripheral proteins or other components
Membrane protein mobility and distribution may be restricted to specific domains (regions) of the cells plasma membrane
Example:
Intestinal epithelial cell
Mammalian sperm cells
29. VI. B. 3. Membrane Carbohydrates Glycocalyx
Many integral membrane proteins have carbohydrate groups attached to their exterior domains
This carbohydrate, together with carbohydrate attached to phospholipid molecules, forms the glycocalyx
Functions
Cell adhesion
Cell recognition
30. C. Diffusion and Active Transport Simple Diffusion
Movement of substances directly across a phospholipid bilayer, with no need for a transport protein
Movement from high ? low concentration
No energy expenditure (e.g. ATP) from cell
31. C. Diffusion and Active Transport Facilitated Diffusion
Movement of substances across a membrane with the assistance of a transport protein
Movement from high ? low concentration
No energy expenditure (e.g. ATP) from cell
Two mechanisms: Channel & Carrier Proteins
Carrier proteins may be uniporters, symporters, or antiporters
32. C. Diffusion and Active Transport Active Transport
Movement of substances across a membrane with the assistance of a transport protein
Movement from low ? high concentration
Energy expenditure (e.g. ATP or ion gradients) from cell
Active transport pumps are usually carrier proteins
Examples:
Na+ K+ ATPase pump
Glucose co-transport pump
33. C. Diffusion and Active Transport Active Transport (cont.)
Active transport ATPase pumps are divided into three types:
P-type pumps phosphorylate themselves during their cycle and include the plasma membrane Na+-K+ pump, the sarcoplasmic reticulum Ca2+ pump, and many other ion pumps
ABC transporters primarily pump small molecules rather than ions; these are the largest family of membrane transport proteins. Each member of this family contains two highly conserved ATP binding sites
34. C. Diffusion and Active Transport Active Transport (cont.)
F-type pumps are H+ driven ATP synthases in mitochondria and chloroplasts; also are similar to V-type pumps that use ATP to pump H+ into organelles such as lysosomes
35. D. Electrical Properties of Membranes Gated Ion Channels:
Voltage-gated, mechanically-gated, or ligand-gated
Example: Voltage-gated K+ channel
Ion channels are usually selective; the best understood is the bacterial K+ channel
36. D. Electrical Properties ofMembranes Events in a Nerve Impulse
The resting potential of a nerve cell: The exterior of the cell is positively charged, due to the gradients of Na+ and K+. (these are maintained by K+ leak channels and Na+-K+ ATPase)
In the resting state, the voltage-gated Na+ channels are closed.
A nerve impulse is a wave of depolarization along the neuron, caused by the Na+ channels opening and Na+ rushing into the cell.
37. D. Electrical Properties ofMembranes Events in a Nerve Impulse (cont.)
Within a millisecond after a nerve impulse passes a section in a neuron, the Na+ channel goes into an inactive state until the membrane is repolarized, when it returns to the closed configuration
A voltage-gated, delayed K+ channel opens and lets K+ rush outside the cell. This, together with the action of the Na+-K+ pump, returns the region to its resting potential.
38. D. Electrical Properties ofMembranes Events in a Nerve Impulse (cont.)
When a nerve impulse reaches the end of a neuron, it triggers the release of neurotransmitter molecules (e.g., acetylcholine or glutamate) from vesicles within the cell.
The neurotransmitter diffuses across the synapse, activates ligand-gated Na+ channels in the next neuron to start the wave of depolarization in the next neuron.