The superfamily of small GTP-binding proteins (GTPases) comprises more than 150 structurally related proteins that perform regulatory functions in cells. Many GTPases function as "molecular switches" by cycling between two conformational forms - a GTP-bound "active" state and a GDP- bound "inactive". Guanosine triphosphate (GTP) plays a significant role in energy transfer and activation of substrates in metabolic reactions. Diseases such as diabetes, allergies, depression, cardiovascular defects and cancer are associated with derangement of G protein signaling. Increasing interest to the regulatory mechanisms of the GTP-binding proteins requires novel assay methods to reveal biochemical mechanisms. Our study focuses on development of lanthanide luminescence based tools for GTPase investigation.
This study aims to develop three assay formats to investigate GTPase function: 1) exchange of small GTPase bound GDP to labeled non-hydrolyzable GTPγ for guanine nucleotide exchange factors studies, 2) hydrolysis of small GTPase bound labeled hydrolysable GTPγ for investigation of GTPase activating proteins, and 3) membrane-based small GTPase Ras assay for allosteric modulator studies. The project is a fundamental study where novel types of homogeneous assay formats for GTP-related research are investigated and developed to reach further understanding on GTPase structural and mechanistical aspects.