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H Higher 3



Paper 1 19 September 2012
2 hour 30 minutes
Additional materials: Answer Paper


Write your index number, CT group & name on all the work you hand in.
Write in dark blue or black pen on both sides of the paper.
You may use a soft pencil for any diagrams, graphs or rough working.
Do not use staples, paper clips, highlighters, glue or correction fluid.

Section A
Answer all questions.
Section B
Answer three out of four questions.
Section C
Answer the question

At the end of the examination, fasten all your work securely together. Please hand in this question
booklet together with your answer sheets.

The number of marks is given in brackets [ ] at the end of each question or part question.
You may use a calculator.
You are reminded for the need for clear presentation in your answers.

This document consists of 8 printed pages.

Raffles Institution
Internal Examination

2012 Preliminary Examination 9815/01

2 Examiners

Section A

Answer all the questions in this section.

1 The Rab family forms the largest branch of the Ras superfamily of GTPases, acting as
essential regulators of membrane traffic pathways.

(a) Explain what is meant by the term family. [2]

(b) Discuss the benefits and limitations of studying protein families compared with the study of
DNA sequence. [4]

(c) Describe the significance of transferrin in iron transport. [2]

(d) Rab11 and Rab7 are located in recycling endosomes and late endosomes respectively.
Describe an experiment to investigate the fate of transferrin after entering cultured
hepatocytes (liver cells). State a limitation of the technique used in your proposed
experiment. [4]

2 Human zona occludens 1 (ZO-1), is a 220 kDa tight junction protein that belongs to the
membrane-associated guanylate kinase (MAGUK) family. Members of this family are found at
the epithelial and endothelial intercellular junctions.

Two different isoforms of ZO-1, (+) and (-) exist as a result of alternative splicing.
(+) is 220kDa and contains an 80-amino acid motif that is absent in (-).

In a research project to determine the distribution of (+) and (-) in guinea pig sertoli cell,
tissue homogenate from adult guinea pig kidney (k), seminiferous tubules (st) and interstitial
cells (ic) were subjected to western blot analysis using Ab #7446, a mouse monoclonal
antibody raised against ZO-1. A photograph of the western blot is shown in Figure 2.1 below.

Figure 2.1

(a) Describe briefly how ZO1 protein was extracted for western blot analysis. [2]

(b) Describe the principles of the western blot technique that was used to analyse ZO-1. [4]

(c) On the diagram, identify the ZO-1 isoforms. Explain your answer. [2]

(d) Discuss the results shown in Figure 2.1. [2]

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3 Examiners

A further experiment was performed to study the expression of the ZO1 isoforms in a
homogenate of seminiferous tubules from 14-day old (y) and adult (a) guinea pig testes. A
western blot analysis was conducted using antibodies against specific ZO-1 isoforms and the
results are shown in Figure 2.2 below.

Figure 2.2

(e) Explain the specificity of the antibody against (+) but not (-). [2]

3 Crystallization of proteins is the rate-limiting step in structural proteomics. In studying the 3D

structure of proteins found in Methanobacterium thermoautotrophicum via X-ray
crystallography, the researchers omitted membrane proteins (about 30% of the proteome) and
any proteins which had obvious homologues in the Protein Data Bank (PDB), (about 27% of
the proteome).

(a) (i) Explain the reason for the omission of the membrane proteins. [1]

(ii) Suggest a reason for the omission of proteins which had obvious homologues in the
PDB in this X-ray crystallographic study of protein 3D structure. [1]

(iii) If the scientists found proteins with homologues in the PDB, what else could they do
to gather additional information on these protein sequences? [1]

(b) The goal of crystallization is usually to produce a well-ordered crystal that is large enough
to provide a diffraction pattern when hit with X-rays. Why is crystallization of proteins
difficult? [2]

(c) Most folded proteins are marginally stable in water at room temperature. A folded protein
is actually a thermodynamic compromise. Briefly explain this statement. [2]

The study of the 3D structure of aquaporin revealed it to be a homotetramer with an hour-

glass shaped pore, with the narrowest constriction to be about 3.0 wide.

(d) Explain briefly how water (H2O) could pass through the pore when protons (H 3O+) are
prevented from doing so. [3]

(e) Based on the hypothesized mechanism of preventing protons from passing through the
pore, predict 2 amino acids that should be conserved in the aquaporin molecules of every
species. [2]

(f) Why is it important that protons are not allowed to pass through the membranes with
water molecules? [1]

2012 Preliminary Examination 9815/01

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4 Examiners

4 FMR-1 is a protein that is associated with Fragile X mental retardation syndrome. The
nucleotide sequence has previously been determined. However, its biochemical function,
intracellular location and 3-dimensional structure has yet to be determined.

(a) Column chromatography is frequently used to purify proteins for downstream analysis.
Describe how antibodies and column chromatography can be used to purify FMR-1. [3]

(b) Following purification, ELISA was used to quantitate the concentration of FMR-1 in eluent
fraction. Describe the technique. [4]

(c) Immunotechniques described in part (a) requires the use of antibodies raised against
FMR-1. However, there are currently no available antibodies that bind to FMR-1 and
previous attempts to raised antibodies against FMR-1 have been unsuccessful.

Suggest how this problem can be overcome using epitope tagging and give details on the
approach. [4]

(d) Green fluorescent protein (GFP) gene can be fused to FMR-1 gene to produce a GFP-
FMR-1 fusion protein in transfected cells. This enables the localization of the fusion
protein to be investigated under a fluorescence microscope.

Name one advantage and one disadvantage of using GFP-FMR-1 protein to investigate
the localization of FMR-1. [2]

2012 Preliminary Examination 9815/01

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5 Examiners

Section B

Answer 3 out of the 4 questions in this section.

5 Postsynaptic density protein 95 (PSD-95) is a membrane associated membrane protein that is

located at the post-synaptic membrane. PSD-95 associates with cytoskeletal elements at
synapses and its overexpression in hippocampal neuron enhances postsynaptic clustering
and activity of glutamate receptor.

Analysis of PSD-95 reveals that it contains 1 PDZ domain, a 90-amino acid protein-protein
binding domains that recognize at least a 3-residue peptide motif in the COOH termini of their
binding partners. This enabled PSD-95 to interact with other proteins to form multimeric
scaffold for clustering of receptors, ion changes and associated signaling protein.

(a) With the help of a diagram, describe briefly how yeast-2-hybrid can be used to identify
protein that interacts with the PDZ domain of PSD-95. [4]

(b) An unknown protein X which interacts with PSD-95 was identified using the technique
described in (a). Protein X was overexpressed and then purified using affinity

Describe how the amino acid sequence of this protein can be determined using Edman
Degradation. [3]

(c) State one limitation of Edman Degradation in sequencing proteins. [1]

(d) The purified interacting partner protein was partially sequenced and its amino acid
sequence is shown below :


Suggest how the protein sequence obtained from part (d) can be further analysed using in
silico methods to predict its function. [2]

6 You have discovered a new protein in macrophages (a type of white blood cell) and have
made some progress into its characterization. In first trying to purify the protein, you found
that it could only be extracted after treating the macrophages with a strong detergent such as
SDS. After purifying the protein you sent it away for automated amino acid sequencing
procedures. The report from the company showed that your protein is composed of 340
amino acids. A partial amino terminal sequence is shown below:


A partial carboxy terminal sequence is shown here:

- Arg-Arg-Gly-Ser-Phe-Gly-Leu-Ile-Leu-Met-Leu-Ala-Lys-Leu-Ala-Gly-Ala-Leu

An ultracentrifugation experiment yields a molecular weight for the protein of 42000 daltons
(Da). The average mass of an amino acid is about 110 Da.

You plug the entire sequence into a computer program that produces a hydropathy plot for
your protein (A hydropathy plot is a graph which shows how hydrophobic each amino acid in a
polypeptide is versus where it is located on the polypeptide. Hydrophobic regions (shaded)
are shown as positive numbers on this type of plot). The plot is shown in Figure 6.1 below.

2012 Preliminary Examination 9815/01

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6 Examiners

Figure 6.1

(a) Using all the information given above, what can you tell about the protein thus far (e.g. its
location, structure, composition etc)? [3]

Your curiosity is greatly aroused. You decide to do an experiment involving radioactive

phosphate (32P). In a special protocol that you have devised, you can incorporate radio-
labeled trinucleotides (i.e. 32PATP, 32PCTP, 32PGTP) into the cytosol of the
macrophages. You run three different experiments. All of these experiments are done in
cell media that contains various nutrients, hormones, and growth factors (but no

(Experiment 1) You add radioactive ATP to the cells along with non-radioactive CTP and
GTP. You find that, after this treatment, your protein becomes radioactive.

(Experiment 2) You add radioactive CTP and GTP along with nonradioactive ATP and find
that your protein does not incorporate any radioactivity.

(Experiment 3) You add radioactive ATP but, beforehand, you completely deplete the cells
of both GTP and CTP. You find that your protein does not become radioactive.

You think you may have an idea of what is happening in the system. You decide to do one
more experiment.

(Experiment 4) You deplete the cells of all CTP, GTP and ATP. You then add back
radioactive ATP, non-radioactive GTP, and a potent, specific inhibitor of adenylate cyclase.
Once again, you find no incorporated radioactivity in your protein. But, when you also add
non-radioactive dibutyryl cAMP (a derivative of cAMP that hydrolyzes to cAMP once inside
the macrophage) you do recover radioactive protein!

(b) Suggest a function for your protein, stating any information that you may have used to
draw this conclusion. [2]

(c) How is your protein becoming radioactive (i.e. how is it incorporating 32P)? Using your
biological knowledge, briefly explain what could be happening in the macrophages such
that it is consistent with the information gained thus far. [5]

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7 (a) Describe the chemical bond that links two amino acids together. [4]

(b) Alpha helix can be predicted more accurately than beta sheet from amino acid
sequences. Based on your knowledge of their structures, explain why this is possible. [3]

(c) Immunoglobulins are multi-subunit proteins. With reference to immunoglobulin G (IgG),

explain which level of protein structure IgG has and describe its antigen-binding site. [3]

8 Carcinogenesis is a multi-step process that frequently results from multiple mutations in

various genes associated with regulation of cell cycle. Gene mutations frequently results in
an altered proteome that leads to dysregulation of the cell cycle, resulting in uncontrolled cell

(a) Describe the principle of a named technique that enables the analysis of the protein profile
of cancerous cell by molecular size only. [3]

(b) Describe a named technique that can be used to analyse the differences in the proteome
of normal and cancerous tissue. [5]

(c) Discuss the advantages of this technique over the technique described in part (a). [2]

Section C

Answer the question in this section.

9 (a) (i) Using aspartate transcarbamoylase (ATCase) as an example, discuss the idea of
allosteric changes in protein conformation produced by ligand binding. [9]

(ii) Briefly explain how you might carry out crystallographic studies to show that there are
allosteric changes in the structure of ATCase during catalysis. [4]

(b) Over the past decade, some researchers have discovered that under physiological
conditions several proteins and protein domains exist with little or no ordered structure.
These proteins are referred to as natively disordered proteins. They have the ability to
undergo disorder-to-order transition upon functioning.

(i) Suggest one advantage and one problem these natively disordered proteins have in
the function of regulation and binding of diverse ligands over natively folded proteins.

(ii) When a globular protein in an aqueous solution starts to unfold and assume a random
coil structure, the entropy of this system changes.

Account for the change in entropy for this system and discuss how some conditions
can disrupt native ordered structure of the protein. [5]

End of Paper
2012 Preliminary Examination 9815/01


List of Amino Acids

2012 Preliminary Examination 9815/01