Download 4c-Observation and Analysis of Cytochrome Proteins Various

Created by D. Norton 2009. Bioinformatics Follow upActivity to LAB- The Effect of Cigarette Smoke on Oxidative Phosphorylation in
Yeast , adapted from AP Biology Lab Series #4 and Effect of Cigarette Smoke on Oxidative Phosphorylation in Yeast, D. Koehnen/Rushford
The Effect of Cigarette Smoke on Oxidative Phosphorylation in Yeast- Observation of
Mitochondrial Cyctochrome Complex Proteins in Yeast and Humans and Amino Acid
Sequence Sequence Comparison in Yeast, Humans and Cows!
I. Introduction
Cigarette toxins are documented to interrupt the activity of the cytochrome proteins in the
mitochondrial electron transport pathway. [Journal of Biological Chemistry vol 217, page 453] This
experiment looks at the effect of cigarette smoke on the activity of electron transport in yeast
cells. It is an adaptation from AP Biology Lab #4B [Measuring Photosynthetic Activity in Spinach Tissue,
Using Spectrophotometer Analysis].
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II. Objective: In the lab, observation of inhibition of the yeast mitochondrial electron
transport mechanism [functionality as measured with ability of electrons to reduce DPIP
indicator, using quantitative %transmittance analysis for the rate of DPIP reduction] was
observed using normal [healthy] yeast and yeast exposed to both filtered and nonfiltered
cigarette smoke [aspirated into water samples]. In this activity, the structure of one of the
cytochrome protein complexes will be viewed and compared between yeast and human
chytochrome variants, using Cn3D4.1 protein viewer software at the NCBI site. This
software is a free download from NCBI. In addition, the sequence variations between yeast,
humans and >>>> [an organism closer to humans in evolutionary descent] will be viewed to
identify the relationship between time of evolutionary “split” and sequence variation.
*Effect of Cigarette Smoke on Yeast Respiration
Mitochondrial respiratory chain/cytochrome
chain/cytochrome complexes
The mitochondrial respiratory chain is
composed of a number of large multisubunit complexes that are embedded in
the inner mitochondrial membrane.
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Created by D. Norton 2009. Bioinformatics Follow upActivity to LAB- The Effect of Cigarette Smoke on Oxidative Phosphorylation in
Yeast , adapted from AP Biology Lab Series #4 and Effect of Cigarette Smoke on Oxidative Phosphorylation in Yeast, D. Koehnen/Rushford
III. Using NCBI to access and compare cytochrome b1 protein structures.
1. Enter the NCBI site here: http://www.ncbi.nlm.nih.gov/
2. At the top center of the menu bar, use the search drag down menu to select for
structure 1KB9. This will bring up the yeast cytochrome b protein structure.
3. Click on the image of the protein. This will bring you to the protein structure window.
4. Click on structure view in Cn3D just below the image. [If you don’t already have
Cn3D4.1 protein viewer software downloaded, you can do this with the download
button just below the structure view in Cn3D button.] Your viewer should launch in a
new window.
5. You should see a protein structure in the top, with a window with amino acid
sequences below. You can use your mouse to drag the protein structure around to
view it from several perspectives. Reduce these windows to ½ of your computer
screen.
6. Go back to your NCBI site window and in the upper right/search window, enter
1NTM, the human cytochrome c protein structure. When the site opens for this protein
structure, again hit the button structure view in Cn3D just below the image to open the
human protein. Also reduce these views to ½ computer screen view, so that you can
see both the yeast and human protein structures side by side, along with their protein
sequences below. See an example view here:
http://www.genetics.org/cgi/reprint/125/1/9
1NTM
1KB9
7. In the sequence viewer below, use your mouse to select/highlight the first/”A” amino
acid sequence. You will need to use your control key to select all of the sequence, as
you scroll across the bottom of the screen to move right. As you are highlighting this
sequence [yellow], view the protein above; the part of the protein that corresponds to
this protein subunit should be changing to a yellow highlight also.
8. Go to your human protein view and highlight the same protein subunit/”A” sequence.
9. Observe the area of the human protein that is highlighting yellow in the upper view.
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Created by D. Norton 2009. Bioinformatics Follow upActivity to LAB- The Effect of Cigarette Smoke on Oxidative Phosphorylation in
Yeast , adapted from AP Biology Lab Series #4 and Effect of Cigarette Smoke on Oxidative Phosphorylation in Yeast, D. Koehnen/Rushford
http://www.genetics.org/cgi/reprint/125/1/9
1KB9
1NTM
10. What do you notice about the yeast and human cytochrome proteins for sequence
“A”? They code for the same region.
11. Now observe 30 amino acids from the A polypeptides of the yeast and human
samples, along with the same region from a cow. This has been selected for you, in
interest of time, but you can select these regions from the Cn3D4.1 Viewer, if
preferred.
tatyalqsvpetqvsqldnglrvaseqs
aevtqlsngivvatehnsahtasvgvv
tatyaqalqsvpetqvsqldnglrvase
human
yeast
cow
12. What do you observe about the amino acid sequence of the yeast compared to the
human/cow samples? Significantly different, but all code a functional cytochrome Bc1 complex.
13. Would you expect these sequences to be more similar for the cow and human than for
either of these two in comparison to the yeast? Why? Cow/humans have a more close relationship in
evolutionary past; both are in the animal branch, whereas the animals and fungi split much earlier in time and have therefore
had a greater amount of time to accumulate mutations/differences.
VI.
Conclusion:
From LAB: What is the effect of cigarette smoke on oxidative phosphorylation
[electron transport within the cytochrome complexes of the mitochondria] in
yeast? Yeast respiration is reduced when organisms are exposed to filtered and
non-filtered cigarette smoke.
Does this effect on yeast merit any consideration for consideration in humans?
Explain.
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Created by D. Norton 2009. Bioinformatics Follow upActivity to LAB- The Effect of Cigarette Smoke on Oxidative Phosphorylation in
Yeast , adapted from AP Biology Lab Series #4 and Effect of Cigarette Smoke on Oxidative Phosphorylation in Yeast, D. Koehnen/Rushford
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