Reference no: EM133189440
SECTION A
Part A
Question 1
1. This question relates to a viral proton channel essential for viral replication. DATA
A. This channel is a tetramer, with each subunit containing 97 amino acids and a molecular weight of 11.2 kDa. The amino acid sequence of the monomer is shown in Fig. 1.
Figure 1 - Amino acid sequence
B. Hydropathy analysis of the protein reveals a number of hydrophobic residues. The hydropathy plot from the amino acid sequence in Figure 1 is shown in Figure 2. The plot is based on the Kyte-Doolittle scale with a window of 9 residues.
C. This is a pH gated channel, with its proton flux activated at low pH. The gating mechanism relies on conformational changes on Trp41 resulting from protonation changes on H37.
D. The helical wheel plot for residues L26VVAA SIIGILHLIL WILD" results in an average hydrophobicity of 0.370 and a magnetic moment of 0.132 (Figure 3A). The helical wheel plot for residues R45LFFKC IYRFFEHGLKRG62 results in a magnetic moment of 0.406 and an average hydrophobicity of -0.216 (Figure 3B)
E. The proton channel has been purified in a range of detergents (named A, B, C and D) and then reconstituted into liposomes. The proton channel activity has been recorded for these four samples (Figure 4).
F. Labeling experiments with the channel inhibitor Amantadine have identified a high affinity site between the residues V27-S31. The binding affinity of Amantadine has then been measured in a range of detergents (named A, B, C and D) and in liposomes and the Kds are shown in Table 1.
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Binding Affinity (nM)
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Liposomes
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100
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A
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20
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B
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120
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C
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500
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D
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no binding
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Questions
a) Using all the available data, how many transmembrane helices and other membrane interacting regions are predicted? Indicate the locations (amino acids numbers) of the transmembrane domains and any other membrane interacting regions and draw a topological map of the proton channel showing all the membrane interacting segments, N- and C-termini and regions involved in Amantadine binding. Briefly explain how you came to this answer.
b) Using the proton flux data (Figure 4), Discuss which of these detergents retain more of the native-like properties of the proton channel. Briefly justify your answer.
c) Based on the Amantadine binding data, discuss the suitability of each detergent and justify the possible effect of each detergent in this assay.
d) The proton channel activity can be modulated by the lipid composition. Based on the amino acid composition of the membrane interacting regions, which type of lipids will have stabilizing and destabilizing effects and why?
e) New strains of the virus show reduced effects of the antiviral drug Amantadine. Propose a hypothesis that explain this effect.
SECTION B
Answer ONE question from Section B
Question 2
Compare and contrast the fluid mosaic model first proposed by Singer and Nicholson in 1972 with the modern fluid mosaic membrane model.
Question 3
Describe how specific lipid molecules can alter the properties of certain proteins. Discuss how the cell make use of this process to modulate specific cellular functions.
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