A novel method to characterize interactions among biomolecules in living cells

STORTZ, M; PRESMAN DM; POOLEY, JR; HAGER, G; PECCI, A.; LEVI V

Puerto Varas

Congreso; 1. XII PABMB Congress.; 2013

SAIB

Studying interaction among biomolecules is a relevant issue in biology. Proteins form homo- and hetero-complexes, with functional relevance in cell. There are many techniques that allow to study the physicochemical features of these interactions in vitro, but this characterization could may not refflect what happens inside the cell. Other techniques allow to study protein interactions in vivo, like co-immunoprecipitation assays, but are mostly qualitative. Here we present a method based on an advanced fluorescence microscopy technique, that allows to quantitatively study interactions between biomolecules in living cells, the Number and Brightness (N&B) assay. The 1-color N&B assay provides information about homo-complexes formation. Here we present the development of a 2-color N&B assay to study interactions between two different kind of molecules, i.e. formation of hetero-complexes. This method makes possible to determine the complex stoichiometry (CS) and explore the complex formation constant (Kf). Previously, we have studied the mechanisms involved in the ligand-dependent homo-dimerization of the glucocorticoid receptor (GR) by using 1-color N&B. The activated GR translocates from the cytoplasm to the nucleus and there exerts its role as a transcription factor, with positive or negative regulation of gene expression. This regulation involves the interaction of the GR with DNA and/or another transcription factors and co-regulators. Our goal is to study these interactions to understand the mechanisms involved in GR activity.