Substrate targeting mechanism of a DesCEND pathway

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Abstract This request for a supplement to the grant R01GM135592 proposes the acquisition of an automated high- throughput crystallization screening system to support the research aims that focus on investigating the substrate targeting mechanisms of the F-box subunit FBXO31 of the SCF ubiquitin ligase in a novel DesCEND pathway. By identifying and characterizing substrates of FBXO31 at both a systems level and the molecular level, we proposed to elucidate the complementary structural determinants of the FBXO31–degron pair that confer substrate specificity and dictate protein recognition and fate. To achieve our goals, it is vital that we have the ability to screen wide ranges of crystallization conditions that are essential for obtaining high resolution protein- substrate complex structures. This proposal requests funds to replace a 10-year old Gryphon-100ul Flex instrument with a similar but up-to-date version from the same manufacturer. In addition to having reputable performance quality, the proposed system covers a broad range of screening applications and meets the automation requirements needed to develop a streamlined approach for crystallization in a high-throughput fashion. We have obtained institutional support from the Department of Molecular Biology and Biophysics. The instrument will ensure the continued biochemical, biophysical and structural studies proposed in the parent award.