Session 2.4a - Group Work - Lecture 1

Question 1

Membrane Phospholipids and Cholesterol

1) Modes of Mobility for Phospholipids

There are four modes of mobility allowed for membrane phospholipids.

a) Briefly describe each of these below What are they?

Answer 1

1. Flexion - intra-chain motion which is a kink formation in the fatty acyl chains
2. Rotation - fast axial rotation around its axis
3. Lateral diffusion - fast lateral diffusion within the plane of the bilayer
4. Flip flop - movement of lipid molecules from one half of the bilayer to the other on a one for one exchange basis. This is rare.

These all have increasing energy

Question 2

1) There are four modes of mobility allowed for membrane phospholipids.

b) Based on your answers draw a simple diagram illustrating the
four modes

Answer 2

See Slide 18 Lecture 1

Flexion/intra-axial motion - kink in leg

Rotation - around a singular axis

Lateral diffusion - diffusion along the plane

Flip-flop - swapping with another phospholipid on the opposite bilayer on a 1:1 basis.

Question 3

Effect of Temperature on Modes of Phospholipid Mobility.

2) a) Thinking about last week’s session on Homeostasis and thermoregulation - what do you think the relationship is between these four modes of movement and changes in temperature in the internal environment or milieu interieur of the body ?

Answer 3

The more the temperature is increased, the more energy the bilayer has. This means that movement is going to be more permitted - so higher movements such as the ‘flip flop’ motion are more likely to occur.

Question 4

2) Thinking about last week’s session on Homeostasis and thermoregulation - what do you think the relationship is between these four modes of movement and changes in temperature in the internal environment or milieu interieur of the body ?
b) Is this physiologically relevant to the function of the membrane ?

Answer 4

This is physiologically relevant because the membrane is fluid/dynamic, so the more energy it has the more movement it can produce, which is vital for its function, as things in the membrane need to move to get places.

Similarly, facilitated diffusion occurs faster at higher temperatures, so increasing the temperature increases the membrane permeability.

Question 5

Composition of Membranes 1

3) Both membrane phospholipids and proteins are said to be amphipathic.
a) What does amphipathic mean?

Answer 5

A molecule which contains both a hydrophilic and a hydrophobic moiety

Question 6

3) Both membrane phospholipids and proteins are said to be amphipathic.
b) Draw a simple diagram of a phospholipid and label the parts described by the term amphipathic.

Answer 6

See Slide 8 for drawing

Phosphate and head group = hydrophilic

Fatty acids = hydrophobic

Question 7

Composition of Membranes II

4) The precise composition of specific membranes varies according to cell and tissue type.
a) In general what is the dry weight composition of the main components of cell membranes?

Answer 7

40% lipid
60% protein
1-10% carbohydrate

Question 8

4) The precise composition of specific membranes varies according to cell and tissue type.

b) The components by dry weight do not include an essential component. What is the component in living cells and what is the proportion of this
component in a living cell ?

Answer 8

Water

20% of total weight

Question 9

4) The precise composition of specific membranes varies according to cell and tissue type.
c) How important is this component [essential total weight component not included in dry weight] to normal physiological function of the membrane ?

Answer 9

Water is important for the physiological function of the membrane, because it is water which creates the orientation of the membrane bilayer - the hydrophobic parts of the bilayer face inwards whilst the hydrophilic heads face outwards, touching water

Question 10

4) The precise composition of specific membranes varies according to cell and tissue type.
d) Think about last week’s session on Homeostasis and keeping certain physicochemical attributes within tight limits. In general terms what might happen to membrane structure and function if the concentration of this component [the essential non-dry weight component] was reduced by just a few percent ?

Answer 10

The shape of the membrane would be affected if there was less water - this would mean the membrane would have to be closer together which would have an impact on the function of the cell/organelle and the membrane itself.

The cell would be dehydrated (hypertonic) thus the membrane would shrink/be compressed. This would disrupt the cell membrane, so normal passive diffusion will also be affected as well as any other proteins involved.

Just need to know membrane shape is affected but not how

Question 11

Composition of Membranes III

5) a) What is the range of length by carbon groups of fatty acid chains in a phospholipid.

Answer 11

14 - 24 carbons

Lengths carbon 16-18 is most prevalent

Question 12

5) b) For phospholipids, with unsaturated fatty acid chains how do you think mobility varies with length?

Answer 12

Differences in the length and saturation of the fatty acid tails are important because they influence the ability of phospholipid molecules to pack against one another, thereby affecting the fluidity of the membrane

?
An increase in unsaturated fatty acid length either means increased mobility because of less phospholipid packing, or a decrease in mobility because it requires less energy to move it?

If we are talking mobility for the individual phospholipid, the shorter the length the increase in mobility as less energy is required to move it.

• Mobility = individual phospholipid, whereas fluidity = whole plasma membrane. Therefore an increase in unsaturated fatty acid length means a decrease in MOBILITY, bc more energy is required to move it.

Question 13

5) c) Draw a simple diagram of (i) a phospholipid with saturated and (ii) an unsaturated fatty acid hydrocarbon chain. What is the effect of having phospholipids with unsaturated fatty acid chain on membrane fluidity? How is this related to the unsaturated structure?

Answer 13

See Fig. 19

i) Straight (no double bond)
ii) Kinked (double bond)

Membrane fluidity = the whole plasma membrane, not individual phospholipid.

Unsaturated fatty acids increase membrane fluidity. This is due to their ‘kink’ in the structure, which reduces phospholipid packing, thus the increase in fluidity.

Question 14

Measuring Phospholipid Mobility in the Membrane

6) Experiments have shown it may take up to one day for a phospholipid to move from one lamella of a lipid bilayer to the other. By contrast the same phospholipid molecule may move an equivalent distance i.e about 10 nm, in the plane of the bilayer in as little as 2.5 us.
a) How do you account for this difference in mobility?

Answer 14

A lot more thermodynamic energy is required to move a phospholipid across the lamella of the bilayer.

This is because lateral diffusion occurs in the same plane and does not require crossing of the hydrophobic/hydrophilic moieties.

However, in flip flop motion, the hydrophilic head has to cross the hydrophobic tail of the membrane, which requires a lot of thermodynamic energy. As the phospholipid does not want to do that it will take a long time to gain enough energy required to overcome these interactions.

Question 15

Composition of Membranes - Cholesterol

7) a) Cholesterol is an important component of cell membranes and is structurally very different to that seen in phospholipids. Draw a simple diagram of cholesterol and label its three main structural domains.

Answer 15

See Slide 21 Lecture 1

• Polar head group
• Rigid planar steroid ring structure
• Non-polar hydrocarbon tail (flexible)

Question 16

7) b) What is noteworthy about the hydrophilic group of cholesterol ?

Answer 16

It makes hydrogen bonds with the carbonyl-oxygen of the fatty acids, so it comes and anchors itself alongside every other phospholipid. This homogenises that part of the membrane by abolishing endothermic phase transition

Question 17

7) c) How many carbon ring structures are present in cholesterol ?

Answer 17

4

Question 18

7) d) Draw a simple diagram of the orientation of cholesterol within the phospholipid bilayer.

Within the diagram, label the effect Cholesterol has on the mobility of the adjacent phospholipid. Related to its structure, provide a brief description of why cholesterol has these effects on phospholipid mobility.

Answer 18

See Fig. 24

OH of cholesterol hydrogen bonded to the C=O of fatty acid; cholesterol implanted itself against the phospholipid bilayer.

The rigid sterol ring reduces phospholipid mobility because the phospholipid cannot move around it.

However, the flexible tail of the cholesterol is also reducing packing as the PLs are further away from each other, thus increasing phospholipid mobility.

Question 19

7) e) Because of its interactions with phospholipids, cholesterol is described as having a ‘paradoxical’ effect on overall fluidity of cell membranes.

Briefly describe the aspects of this ‘paradoxical effect’

Answer 19

Reduced phospholipid chain motion, reduced fluidity at the rigid sterol ring portion of cholesterol.

Reduced phospholipid packing, increased fluidity bc cholesterol is packing in between PLs, preventing them forming that hexagonal crystalline array.

So at low temp prevents packing, at high temp reduces motion.

Question 20

Function of Cholesterol in the Plasma Membrane

8) a) Given your answers to the above questions what role does cholesterol play in the plasma membrane of living cells ?

You should consider how cholesterol acts to decrease membrane fluidity at high temperatures and increase fluidity at low temperatures?

Answer 20

Cholesterol acts to abolish the endothermic phase transition and homogenise the membrane properties.

At high temperatures, cholesterol acts to decrease membrane fluidity by packing its structure in between the molecules, so the phospholipid molecules cannot move around its rigid steroid ring. This reduces motion at high temperatures.

At low temperatures, cholesterol increases fluidity by preventing packing of the hexagonal crystalline structure by inserting itself in between the PLs. Therefore, at low temperatures, fluidity is increased bc packing cannot occur as efficiently.

This means cholesterol acts to decrease membrane fluidity at high temperatures and increase fluidity at low temperatures, thus abolishing the endothermic phase transition and stabilising the dynamic properties of the membrane.

Question 21

Interpreting Experimental Data:

Phospholipid Flip-Flop Fun

9) Some scientist have experimentally investigated the rate of transverse diffusion or ‘flip-flopping’ of phospholipids in a bilayer membrane.

This was carried out using a paramagnetic analogue of phosphatidylcholine, called spin-labelled phosphatidylcholine as shown in the accompanying picture in the Workgroup folder.

The nitroxide (NO) group in spin-labelled phosphatidylcholine molecule gives a distinctive paramagnetic resonance spectrum. This is measured using some special equipment known to scientists as a spectrometer.

This paramagnetic NO signal on this analogue molecule disappears when nitroxides are converted into amines by reducing agents, such as ascorbate.

Note ascorbate also more commonly known as Vitamin C is water soluble and will not cross a phospholipid membrane.

a) Experimental Observation 1

Lipid vesicles containing phosphatidylcholine (95%) and the spin-labelled analogue (5%) were prepared by sonication and purified by gel-filtration chromatography. Most importantly, the amount of signal from the paramagnetic resonance spectrum decreased to 50% of its initial high value within a few minutes of the addition of ascorbate or Vitamin C.

What do you think this result indicates ?

Answer 21

50% of the spin-labelled analogue was acted on by ascorbate (Vitamin C) within a few minutes.

This suggests the outer lamellae of the membrane was acted on by ascorbate, whereas the other half of the spin-labelled analogue in the bilayer (facing inwards) was unaffected (Vitamin C is WATER-SOLUBLE and will not cross the membrane).

Question 22

9) Some scientist have experimentally investigated the rate of transverse diffusion or ‘flip-flopping’ of phospholipids in a bilayer membrane.

This was carried out using a paramagnetic analogue of phosphatidylcholine, called spin-labelled phosphatidylcholine as shown in the accompanying picture in the Workgroup folder.

The nitroxide (NO) group in spin-labelled phosphatidylcholine molecule gives a distinctive paramagnetic resonance spectrum. This is measured using some special equipment known to scientists as a spectrometer.

This paramagnetic NO signal on this analogue molecule disappears when nitroxides are converted into amines by reducing agents, such as ascorbate.

Note ascorbate also more commonly known as Vitamin C is water soluble and will not cross a phospholipid membrane.

b) Experimental Observation 2

The remaining paramagnetic signal was recorded for a couple of days by dedicated Ph D students. This signal related to the NO group followed a slow exponential decay with a half-time of 6.5 hours. That means every 6.5 hours it reduced by 50% in magnitude.

This meant that Vitamin C was still acting to reduce NO groups but much more slowly

How would you interpret these changes in the amplitude of the paramagnetic NO signal?

Answer 22

This suggests that it takes 6.5 hours(?) for a phospholipid to flip flop - thus, in each change of flip flop, more phospholipids are being reduced by ascorbate, hence the exponential decay in paramagnetic NO signals.