Pre-Intro Bio: Williams and O’Dowd, UC Irvine

Unit 1 Analysis of Research:

Peter Agre, 2003 Nobel Prize winner for discovery of aquaporins

This Analysis of Research will take you through the speech given by Dr. Peter Agre upon receiving the
Nobel Prize for Chemistry in 2003. His speech summarizes the path of discovery of the water channel.
Not only does it demonstrate how scientific research is conducted, but Dr. Agre emphasizes the
collaborative nature of science by crediting all of his associates.
By the end of this worksheet, you should be able to:
1. Describe the basics of the research techniques used in the four experiments outlined.
2. Solve application level questions associated with the science by using knowledge and concepts
learned in the basics videos.
3. Work collaboratively with classmates by providing thoughtful questions, answers and resources
on the discussion forum

Your main source will be the transcript of the lecture given by Peter Agre:

[Link]
2003/[Link]
You will only use the first five pages for this activity (pg 184-188)
If you have problems accessing this from Safari then use another browser like Chrome or Firefox

Main page: [Link]

The references for the figures used in this worksheet are at the end of this worksheet. Open Source
articles are marked with an “*.” These are not needed to complete the worksheet.
Pre-Intro Bio: Williams and O’Dowd, UC Irvine

Part 1: Introduction
Dr. Agre starts his lecture by describing work that came before his.
1. What cell types were known to have high water permeability before aquaporins were discovered?

2. Explain in your own words Agre’s logic for proposing why there must be a selective water channel
in at least some cells.

Part 2: An Accidental Discovery

Dr. Agre discussed how they accidentally found a 28 kDa protein while trying to better understand
another membrane protein, rH factor, in red blood cells. Their two main techniques were gel
electrophoresis (SDS PAGE) and immunoblotting (or Western blots). See the end of this booklet for a
description of these techniques and do some research until you have a BASIC understanding of them.
This section will require you to learn some difficult material on your own, so find a quiet space and an
hour to work.

3. What is “kDa” (kilodalton) a measurement of?

4. Why did Agree first miss the presence of aquaporins and instead think they had just found an Rh
molecule?

This figure (from Denker et al. 1988) shows the same proteins in the presence of two stains and
immunoblotting. The arrow and box indicates the location of the “28 kDa” size protein.
Pre-Intro Bio: Williams and O’Dowd, UC Irvine

Lane 1 contains the proteins from whole erythrocyte membranes. The membranes were then washed
with a detergent called Triton X-100 that removes surface proteins but not embedded proteins. Lane 2
contains the treated membrane, Lane 3 contains the proteins that fell off the membrane.

5. Based on these results, is the “28 kDa” protein a protein on the surface of the erythrocyte
membrane or embedded within the erythrocyte membrane?

Once a researcher has made the antibody to a mystery protein, other

tissues can be treated with the labeled antibody to look for the protein.
This image (arrows) shows the stained proteins in black (Denker, 1988).

6. Where else in human tissue did they find the protein?

Part 3: Sequencing the Mystery Protein

In the paper by Preston and Agre (1991), they sequence the mystery protein, now named CHIP28.

7. How many amino acids make up this protein? _______________

Once the amino acid sequence of the protein was established, computer programs can estimate the
shape of the protein and any regions that are nonpolar. This output using the Kyle Doolittle methods
represents hydrophobic regions with values above 0 and hydrophilic regions with values below zero, and
the image shows the computer simulation of the protein shape (from Preston and Agre, 1991).
Pre-Intro Bio: Williams and O’Dowd, UC Irvine

8. Based on this information, what part/s of the protein are likely to be next to the phospholipid tails
in a cell membrane (A,C, E or B, D, or 1,2,3,4,5,6 )? Why?

Part 4: Proof of Water Transport

At this point, Dr. Agre had established that the protein was common enough and the correct shape to be
a water channel. But he needed to show that it did, in fact, transport water. The final two experiments
showed this elegantly.
(From Preston et al., 1992) First, they took frog eggs (oocytes), and injected half of them with water, and
half of them with the RNA that coded for the CHIP28 protein. They waited three days to allow the cells
to make protein from the RNA.

9. Why were frog oocytes a good cell for this experiment?

The control (left) and experimental (right) eggs were then placed in a
hypotonic solution. The top panel is after 30 seconds, Bottom panel after 3
minutes.

10. What happened in the oocytes that received the RNA? Explain why
this result occurred in your own words.

(From Zeidel et al., 1994). Finally, the researchers made liposomes (remember liposomes?) and gave
them different amounts of the CHIP28 protein. Here is an example of their data:
Pre-Intro Bio: Williams and O’Dowd, UC Irvine

The line represents the change in volume of a liposome with zero CHIP28 proteins after the liposome is
placed in a new solution.

11. Is the new solution hypertonic or hypotonic? Why?

12. On the figure above, draw in the expected line for a liposome with 250 CHIP28 proteins, and the
expected line for a liposome with 750 CHIP28 proteins. Label the new lines. Concisely explain your
new figure.

Congratulations! This activity took you through SEVERAL very complicated experiments, and you had to
think about hypotheses and analyze data. These are techniques that are critical for biologists and for
students in biology courses. Thank you for working hard on it!
Pre-Intro Bio: Williams and O’Dowd, UC Irvine

Biotechnology referenced in this worksheet:

SDS-PAGE gels
Overview: Proteins are removed from cells, denatured to form simple polypeptide chains that are
negatively charged, and placed at the top of a gelatin-like matrix called a “gel”. In an electric current the
negatively charged proteins are separated by size. Proteins are stained by a dye, and their size is
determined by how far they travel down the gel when compared to standards of known size.

Agre’s Simplified Technique

1. Break down red blood cells and isolate the proteins in the membrane
2. Mix the proteins with a chemical called SDS, a detergent that when dissolved in water forms
molecules that have a net negative charge. These molecules break apart most of the bonds
forming the secondary and tertiary structure of the proteins reducing these to their primary
structure consisting of a linear chain of polypeptides. The polypeptide chain binds the negatively
charged SDS molecules and all polypeptides in mixture are negatively charged.
3. The denatured protein solution is put at top of an electrophoresis gel, which is very slightly
porous.
4. An electric current is put across the gel with a negative electrode at the top and a positive
electrode at bottom of the gel. All of the denature proteins move toward the positive electrode
since they are negatively [Link] smaller proteins move more rapidly through the tiny holes
in the gel and therefore move further in a given time than larger proteins.
5. The reference “lane” of the gel contains denatured proteins of known size. These will act as the
reference sizes.
6. After turning off the current, the gel is stained with a dye so the bands are visible. Thick bands
have lots of protein, thin bands have a small amount.
7. Dye names are Coomassie Blue or silver stain

Immunoblotting
Overview: Immunoblotting is used to look for a desired protein in a band of protein in a PAGE gel. A gel
is run, the proteins are “blotted” onto a membrane and then exposed to an antibody that binds to a
known protein. If the antibody sticks, its label (color or radioactivity) indicates the presence of the
desired protein.

Agre’s Simplified Technique

1. Break down cells, isolate the desired protein.
2. Inject the protein into live rabbits. The rabbits will make antibodies against the foreign protein.
Blood from the rabbits is then removed and the antibodies are isolated.
3. A new sample that may contain the protein is run through a gel to sort proteins by size. The
proteins are blotted from the gel onto a membrane.
Pre-Intro Bio: Williams and O’Dowd, UC Irvine

4. The membrane is immersed in a solution containing the antibodies. If the desired protein is
present, the antibodies bind to the protein, and only that protein.
5. The antibodies are then labeled with a radioactive or colored label molecule. The membrane is
examined for a reaction, which is present only when the protein is present. Since the
membrane was copied from the gel, smaller labeled proteins will be farther down the gel than
larger proteins.

References

*Denker, B. M., Smith, B. L., Kuhajda, F. P., & Agre, P. (1988). Identification, purification, and partial
characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal
tubules. Journal of Biological Chemistry 263, 15634–15642.

Macey, R. I. & Farmer, R. E. L. (1970) Inhibition of water and solute permeability in human red cells.
Biochemica Biophysica Acta 211, 104–106.

*Preston, G. M. & Agre, P. (1991). Isolation of the cDNA for erythrocyte integral membrane protein of 28
kilodaltons: member of an ancient channel family. Proceedings of the National Academy of
Sciences of the USA 88, 11110–11114.

Preston, G. M., Carroll, T. P., Guggino, W. B., & Agre, P. (1992). Appearance of water channels in
Xenopus oocytes expressing red cell CHIP28 protein. Science 256, 385–387.

Zeidel, M. L., Nielsen, S., Smith, B. L., Ambudkar, S. V., Maunsbach, A. B., & Agre, P. (1994).
Ultrastructure, pharmacologic inhibition, and transport selectivity of aquaporin channel-forming
integral protein in proteoliposomes. Biochemistry 33, 1606–1615.