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FP5

SPOT-NOSED Report Summary

Project ID: IST-2001-38899
Funded under: FP5-IST
Country: France

Demonstration of the functional response of olfactory receptors in nanosomes deposited on solid supports by surface plasmon resonance

We used Surface Plasmon Resonance technique for monitoring the functional response of the olfactory receptors in the nanosomes once immobilized on solid suports.

The procedure consisted on two steps: in the first step immobilization of the nanosomes on the sensorchip was verified while in the second specific response of the immobilized nanosomes to odorants were monitorized. Previuous test experiments with bovine rhodopsine membrane fraction were performed.

For the verification of the nanosome immobilization the membrane fraction corresponding to yeast cells expressing rat I7 was injected on top of a L1 sensor chip (gold surface covered with dextran modified with lipophilic compounds). Then subsequently the polyclonal anti-I7 antibody was injected. No detection could be obtained, even for highly concentrated membrane fraction deposition. One possible explanation is that the specific IgG is not concentrated enough within the anti-I7 polyclonal antibody.

Even though no response was obtained in this case, the functional response tests proceeded. The straightforward idea would be to inject the odorant solution on the BIAcore sensor chip after immobilisation of the membrane fraction containing the olfactory receptor. However, since the odorants used are so small, their binding to the receptors of the membrane fraction cannot be directly detected. We thus decided to use a procedure set up by Vogel (Bieri et al.) to follow activation of rhodopsin.

Golf departure is indeed detected upon heptanal (5mM) stimulation of the I7 receptor present in the membrane fraction deposited on the L1 sensor chip (0.004mg/ml total protein concentration in membrane fraction deposited), in the presence of 10mM GTP.

In summary, the olfactory receptor is indeed still in its active form in the membrane fraction deposited onto the sensor chip. Golf is still present in the membrane fraction, pre-associated with the olfactory receptor. Stimulation of the olfactory receptor by its odorant ligand induces its conformational change, and thus its interaction with the a subunit (Golf) of the G protein, which dissociates from the trimer. This event is detected by the BIAcore.

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Reported by

NOPA / NOeMI Research Unit
INRA, blg 440
78352 JOUY-EN-JOSAS
France
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