Dielectric spectroscopy based monitoring of the cellular response to G protein coupled receptor/peptide interactions in vivo.

摘要

When exposed to alternating electric fields, composite materials are polarized by various mechanisms, and their dielectric properties present strong frequency dependence. In the technique of dielectric spectroscopy, an alternating electric field is applied to a sample, and the permittivity and conductivity of that sample is measured over a range of frequencies. The information uncovered using dielectric spectroscopy measurements can reveal both morphological and electrical properties of the constituents of the sample at various size scales.;Current methods for the monitoring of protein interactions in vivo rely on tagging the proteins of interest by fluorescent markers. However, it is ideal to be able to monitor proteins activity without perturbing the system under study. Because of its non-invasive nature and ability to probe both the surface properties as well as the internal layers of biological systems without the need for tagging, dielectric spectroscopy has the potential to play a vital role in protein interaction studies.;In this dissertation, the technique of dielectric spectroscopy has been implemented to study the electrical properties of living yeast cells expressing a well known member of the G protein coupled receptor family, the Sterile2 alpha-factor receptor protein (ste2p). The radiofrequency dielectric measurements of the ste2p expressing population of yeast were analyzed with a competent dielectric mixture theory in order to discern the dielectric properties of the constituent regions of the cells. The properties of the various cellular regions were compared between populations of cells which were exposed to the natural binding ligand of ste2p, the alpha-factor, and those which were not. Interestingly, large changes in both the membrane region of the cells, where the ste2p/alpha-factor interaction occurs, as well as the cellular interior dielectric properties were detected after the addition of alpha-factor to the cell suspending medium. A number of different complementary experiments performed on the yeast are presented in an effort to both support and help interpret these dielectric measurements. The results of this large scale study demonstrate the power of dielectric spectroscopy in its ability to non-invasively discern specific and non-specific cellular reactions to an alteration in the cellular external medium.