TUTORIAL-3

1. What is the evolutionary advantage of fish that live in extreme cold temperatures having membranes with a
high proportion of unsaturated fatty acids?

• In cold temperatures, membranes tend to become less fluid and more solid due to reduced kinetic energy of
molecules.
• Increasing unsaturated fatty acid chains in their membranes helps fish maintain fluidity. Unsaturated chains
introduce kinks or bends, preventing tight packing, and requiring a lower temperature to solidify. This
adaptation allows their membranes to remain fluid and functional in cold environments.
 2. The structure of a lipid bilayer is determined by the particular properties of its lipid molecules. What would
happen if:

The hydrocarbon tails were shorter

Phospholipids had only one hydrocarbon tail instead of two? than normal?
• The lipid bilayers formed would be much more
• You would have a detergent. The diameter of the fluid. The bilayers would also be less stable, as the
lipid head would be much larger than that of the shorter hydrocarbon tails would be less
hydrocarbon tail, so that the shape of the molecule hydrophobic, so the forces that drive the formation
would be a cone rather than a cylinder and the of the bilayer would be reduced.
molecules would aggregate to form micelles rather
than bilayers.
The structure of a lipid bilayer is determined by the particular properties of its lipid molecules. What would
happen if:

All the hydrocarbon tails were saturated? All of the hydrocarbon tails were unsaturated?

• The lipid bilayers formed would be much less
fluid. Whereas a normal lipid bilayer has the
viscosity of olive oil, a bilayer made of the same
lipids but with saturated hydrocarbon tails would
have the consistency of bacon fat.
• The lipid bilayers formed would be much more
fluid. Also, because the lipids would pack together
less well, there would be more gaps and the bilayer
would be more permeable to small, water-soluble
molecules.
The structure of a lipid bilayer is determined by the particular properties of its lipid molecules. What would
happen if:

Each phospholipid molecule were covalently linked through the end carbon atom of one of its
hydrocarbon tails to a phospholipid tail in the opposite monolayer?

• The lipid bilayers formed would have virtually unchanged properties. Each lipid molecule would now span the
entire membrane, with one of its two head groups exposed at each surface. Such lipid molecules are found in the
membranes of thermophilic bacteria, which can live at temperatures approaching boiling water. Their bilayers do
not come apart at elevated temperatures, as usual bilayers do, because the original two monolayers are now
covalently linked into a single membrane.
 3. Which of the three 20-amino-acid sequences listed below in the single-letter amino acid code is the most
likely candidate to form a transmembrane region (⍺-helix) of a transmembrane protein? Explain your answer.
You may have to look up an amino acid table if you are not familiar with the one-letter codes and the
properties of the amino acids.

a) ITLIYFGNMSSVTQTILLIS
b) LLLIFFGVMALVIVVILLIA
c) LLKKFFRDMAAVHETILEES

To predict which of the three 20-amino-acid sequences is the most likely candidate to form a transmembrane region (αhelix) in a
transmembrane protein, we need to consider the properties of amino acids and their hydrophobicity. In general, transmembrane regions are
often rich in hydrophobic amino acids, as they interact favorably with the hydrophobic core of the lipid bilayer. Polar or charged amino acids
tend to be less common in transmembrane regions.

• Sequence (b) consists of mainly hydrophobic amino acids such as L (leucine), I (isoleucine), F (phenylalanine), G (glycine), V (valine)
and A (alanine). These amino acids are known for their hydrophobic properties, which make them more likely to interact favorably with the
hydrophobic interior of a lipid bilayer.
• In contrast, sequence A contains many polar amino acids (S, T, N, Q) and sequence C contains many charged amino acids (K, R, H, E, D)
which would be energetically disfavored in the hydrophobic interior
of the lipid bilayer.
4) You have isolated two mutants of a
normally pear-shaped microorganism that
First cell extract After urea wash and
have lost their distinctive shape and are
now round. One of the mutants has a defect centrifugation
in a protein that you call A and the other has
a defect in a protein that you call B. First, Membrane Cytosol Membrane Cytosol
you grind up each type of mutant cell and fraction fraction fraction fraction
normal cells separately and separate their
plasma membranes from their cytoplasms.
You set aside a portion of each of these Normal
A and B No A or B A B
fractions for later testing. Next, you wash cells
the remaining portions of the plasma
membrane fractions with a low Mutant A A B A No A or B
concentration of urea (which will unfold
proteins and disrupt their ability to interact
with other proteins) and centrifuge the Mutant B A B A No A or B
mixture. The membranes and their
constituent proteins form a pellet, and the
proteins liberated from the membranes by
the urea wash remain in the supernatant. • Use these observations to classify membrane proteins A and B in
When you check each of the fractions for terms of the way in which they associate with the plasma membrane.
the presence of A or B, you obtain the
results given in the table below.
 Answer of Q4

1. In normal cell extracts, it is evident that Protein A is an integral membrane protein because it remains
associated with the membrane through all treatments. On the other hand, Protein B is a peripheral membrane
protein as it can be removed from the membrane by urea.
2. In the cell extracts from the mutants with a defect in Protein A, Protein A remains in the membrane, while
Protein B does not. This suggests that the mutation in Protein A affects its interaction with Protein B.
Similarly, the same results are obtained when the mutation affects Protein B, further supporting the idea that
Protein A and Protein B interact.
3. This evidence implies that Protein A and Protein B have a functional interaction. The loss of this interaction,
especially between an integral membrane protein and a protein in the cortex, is more likely to result in a
change in cell shape compared to the loss of an interaction between an integral membrane protein and a
protein on the exterior of the cell.
In summary, the observations indicate that Protein A and Protein B are membrane proteins with distinct roles
and interactions, and the loss of their interaction, particularly within the membrane, has a significant impact on
the shape of the microorganism, highlighting the importance of their interplay in maintaining cell morphology.
5. Sodium dodecyl sulfate (SDS) and Triton X-100 are both detergents that can be used to lyse cells to study individual components. Your first project as a
Ph.D. student is to create an in vitro model that allows you to study the mechanism of action of a novel glucose transporter protein. You use SDS to lyse the
cells. Later experiments demonstrate that the transporter is not functional. Provide a possible explanation for what happened, propose a solution for your
next experiment, and articulate the reasons you think the new method will be more successful.

• The use of Sodium Dodecyl Sulfate (SDS) as a detergent for cell lysis likely denatured and inactivated the
transporter protein. SDS is a potent ionic detergent that not only extracts membrane proteins but unfolds them
completely, rendering them non-functional for further study. To address this issue and improve the
experimental approach, switching to a milder detergent like Triton X-100 is a logical choice.
• Reasons for using Triton X-100:
1. Maintains Protein Structure.
2. Better Preservation of Protein Activity.
3. Less Impact on the Lipid Bilayer.

Triton X-100's suitability for solubilizing membrane proteins makes it a valuable choice for creating an in
vitro model for studying the transporter. Once solubilized with Triton X-100, the active transporter protein
can be incorporated into a new, synthetic membrane bilayer for in vitro studies. This single change in the
detergent choice is expected to yield significantly better results
6. Identify the molecule in each pair that is more likely to diffuse through the lipid bilayer and explain your
answers.
Amino acids or benzene.
• Benzene is a nonpolar, hydrophobic molecule, and
it is more likely to diffuse through the lipid bilayer
because the lipid bilayer is also hydrophobic.
• Amino acids are typically polar or charged and less
likely to diffuse through the lipid bilayer without
the assistance of transport proteins.
Cl- or ethanol
• Ethanol is a small, relatively polar molecule that
can dissolve in the lipid bilayer to some extent
and may diffuse through the lipid bilayer, albeit
more slowly than nonpolar molecules.
• Cl- (chloride ions) is charged and hydrophilic,
making it less likely to diffuse through the lipid
bilayer without the help of ion channels or
transporters.
Identify the molecule in each pair that is more likely to diffuse through the lipid bilayer and explain your
answers.

Glycerol or RNA H2O or O2

• Glycerol is a small, polar molecule, and it
can diffuse through the lipid bilayer
because it is somewhat amphipathic, having
both hydrophobic and hydrophilic
properties.
• RNA is a large, charged molecule that is
hydrophilic and would not be able to
diffuse through the lipid bilayer.
• O2 (oxygen) is a small, nonpolar molecule and is
more likely to diffuse through the lipid bilayer.
• H2O (water) is a highly polar molecule, and while
it can diffuse through the lipid bilayer to some
extent, its diffusion is generally much slower than
that of nonpolar gases like O2.
Identify the molecule in each pair that is more likely to diffuse through the lipid bilayer and explain your
answers.

Adenosine or ATP

• Adenosine is a smaller, less charged molecule and is

more likely to diffuse through the lipid bilayer
compared to ATP.
• ATP (adenosine triphosphate) is a larger, highly
charged molecule, and it would require specific
transport mechanisms to cross the lipid bilayer.
7. If ATP production is blocked in an animal cell, the cell will swell due to an influx of water due to osmosis.
Explain this observation.

• In the absence of ATP production, the Na+-K+ pump ceases to function, leading to an increase in intracellular
Na+ concentration. This disrupts osmotic balance, and the subsequent passive diffusion of water into the cell
due to the increased solute concentration causes the cell to swell.
8. You have prepared lipid vesicles (spherical lipid bilayers) that contain Na+-K+ pumps as the sole membrane protein. All the Na+-K+ pumps are oriented in
such a way that the portion of the molecule that normally faces the cytosol is on the inside of the vesicle and the portion of the molecule that normally faces
the extracellular space is on the outside of the vesicle. Assume that each pump transports one Na+ ion in one direction and one K+ ion in the other direction
during each pumping cycle. Predict what would happen in each of the following conditions:

A. The solutions inside and outside the vesicles contain
both Na+ and K+ ions but no ATP.
• Without any ATP to provide energy for the Na+-
K+pumps, no ions will be pumped. The ATP hydrolysis is
used to drive the conformations change in the transport
protein.
B. The solution outside the vesicles contains both Na + and K+
ions; the solution inside contains both Na+ and K+ ions and ATP.
• The pumps will use the energy from ATP hydrolysis to
transport Na+ out of the vesicles and K+ into the vesicles.
(The pumps will stop working either when the amount of
ATP inside the vesicle is depleted, when the K+ outside
the vesicles is depleted or Na+ inside the vesicles is
depleted.)
8. You have prepared lipid vesicles (spherical lipid bilayers) that contain Na +-K+ pumps as the sole membrane
protein. All the Na+-K+ pumps are oriented in such a way that the portion of the molecule that normally faces the
cytosol is on the inside of the vesicle and the portion of the molecule that normally faces the extracellular space
is on the outside of the vesicle. Assume that each pump transports one Na + ion in one direction and one K+ ion in
the other direction during each pumping cycle. Predict what would happen in each of the following conditions:

C. The solution outside contains Na+; the solution inside

contains Na+ and ATP.
• The pump will bind a molecule of Na+, causing the
ATPase activity to hydrolyze ATP and transfer the
phosphate group onto the pump. A conformational
change will occur, leading to the release of Na+ from
the vesicle. However, because there is no K+ outside
the vesicle, the pump will get stuck at that step, and
subsequent steps of the cycle will not occur.