Project Summary: Arf and Rho small GTPases play critical roles in cellular functions. They cycle the active GTP- and inactive GDP-bound forms. They possess their own unique redox-sensitive motifs. However, the redox regulations and functions of their redox motifs are unknown. Without knowing them, the development of effective therapeutics for diseases associated with them is impractical. The objective of the parental project is to understand the redox-dependent regulations and functions of these proteins. The long-term objective of the parental project is to develop therapeutic interventions for these diseases. This objective includes the identification of novel redox-inert nucleotides that seem to target the redox-sensitive Arf and Rho proteins to block their redox responses. To achieve these goals, the parental grant proposal proposed characterizations of the previously unknown redox-sensitive motifs found in the Arf family of proteins. A multidisciplinary study that uses EPR and fluorescent spectroscopic and mass spectrometric approaches, as well as mutagenesis-based redox biochemistry with novel mechanism-based nucleotide analogs and cell biology methods, was proposed. While the proposed spectroscopic approaches can identify the proposed key intermediates, including thiyl radicals, the predicted resultant formation of the GDP adduct in Arf and Rho GTPases cannot be directly monitored. Also, the method to monitor the formation and decay of the proposed Tyr radical in Rho GTPase was unspecified in the parental grant proposal. Our preliminary studies indicate that the GDP adduct can be detected using UV/Vis spectrophotometric analysis. The Rho Tyr radical can also be monitored by a UV/Vis spectrophotometer. Therefore, we propose to add UV/Vis analysis to the parental grant proposal. Accordingly, we request a fund to obtain a UV/Vis spectrophotometer that is capable of the detection of the GDP adduct and the formation and decay of the Tyr radical. The additional characterizations obtained through mutagenesis-based redox biochemistry and cell biology, as well as other spectroscopic methods, will identify the various unprecedented redox intermediates, allowing one to characterize the regulatory features and redox response properties of the redox-sensitive motifs found in Arf and Rho GTPases. Furthermore, the redox biochemical analysis of this multidisciplinary approach includes the use of nucleotide analogs that are novel redox-inert nucleotides. The UV/Vis spectrophotometer can also monitor changes in the state of novel redox-inert nucleotides in Arf and Rho GTPases. Thus, the use of the UV/Vis spectrophotometer will not only allow an examination of the mechanisms underlying the actions of these redox motifs in the small GTPases, but it will also provide light on a novel therapeutic approach wherein diseases associated with the uncontrolled redox response of these motifs in the small GTPases are reduced or eliminated through redox desensitizati...