Summer Research Program for Science Teachers

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.

1 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

*Effect of Cigarette Smoke on Yeast Respiration Mitochondrial respiratory chain/cytochrome complexes

The mitochondrial respiratory chain is composed of a number of large multi- subunit complexes that are embedded in the inner mitochondrial membrane.

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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:

2 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

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.

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.

3 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 tatyalqsvpetqvsqldnglrvaseqs human aevtqlsngivvatehnsahtasvgvv yeast tatyaqalqsvpetqvsqldnglrvase 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.