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Advanced Cell Biology

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Step 1: Determine which GPCRs are associated with the peach scent. (3.5 pts) Insert the cDNA of all the olfactory GPCRs into E. coli and purify out all the olfactory GPCR proteins. For each GPCR protein, have one control reaction with just the GPCR, and one experimental reaction where the GPCR is incubated with the peach extract. Do this for all the olfactory GPCRs. Run all the samples on a gel. If a compound in the peach extract is the ligand for a specific GPCR, it will cause a conformational change in that GPCR. This will cause the band on the gel to shift relative to the control reaction for that GPCR. This will allow us to identify which GPCRs are associated with the peach scent.

Step 2: Identify a single specific “peach” agonist that can stimulate all of the above determined GPCRs for ultimate incorporation in to a STINK™ Cartridge. Nintendo’s design specifications call for a single compound to be associated with each scent. (4 pts) Find all the chemical compounds present in the peach extract using techniques such as spectroscopy Gather the GPCR proteins found to be associated with peach scent, which were isolated in step 1 For each GPCR protein, have one control reaction with just the GPCR. Then several experimental reactions where the GPCR will be incubated with one of the compounds present in peach extract. Run all of the samples on a gel. Analyze the results as described in step 1. Look for a compound which causes a conformational change in all of the GPCRs associated with peach scent.

There are some drawbacks to Nintendo’s STINK™ delivery system. The machine can only deliver a small single size dose (a “puff”) of each compound. Despite this hardware flaw, Nintendo wishes to be able to control the extent and duration of each scent and they expect PBI to design methods by which to accomplish this fine control. Note that you can deliver a second “puff” of another compound. Consider how you might use each of the following to control the extent and duration of a given scent (0.5 pts each):

GMP-PNP (A non-hydrolyzable analog of GTP)
This non-hydrolyzable analog of GTP will allow extending the duration of the scent. If GTP cannot be hydrolyzed to GDP, Gɑ-GDP and Gβγ would not re-associate, so the signal will not be turned off.

Cholera and/or Pertussis toxins
Cholera toxin locks Gɑs in the GTP bound active conformation, this would extend the duration of the scent. Pertussis toxin locks Gɑi in the GDP bound inactive conformation, this would reduce the duration and extent of the scent.

Inhibitors or Activators of Adenylate Cyclase.
Activators of Adenylate Cyclase would cause production of cAMP, which is a second messenger. This would amplify the signal and increase the extent of the scent. Inhibitors of adenylate cyclase would do the opposite, there would be no and very little cAMP, which would result in little of no amplification of the signal, reducing the extent of the scent.

Inhibitors or Activators of cAMP Phosphodiesterase.
Phosphodiesterase breaks phosphodiester bond and converts cAMP back to AMP, getting rid of second messenger Activators of cAMP phosphodiesterase would get rid of the second messenger and reduce the extent of the scent. Inhibitors of cAMP phosphodiesterase would allow the second messenger to exist and amplify the signal, increasing the extent of the scent.

Inhibitors or Activators of a GRK.
Activators of GRK would cause phosphorylation of serine and threonine residues, which can then bind arrestin proteins, preventing reactivation of the signaling pathway. Activators of GRK would reduce the duration of the scent. Inhibitors of GRK would do the opposite and increase the duration of the scent.

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