PROJECT SUMMARY – ABSTRACT Polycystin ion channel complexes play fundamental roles in human fertilization and embryonic development, although most frequently dysregulation of polycystins leads to renal manifestations. Autosomal dominant polycystic kidney disease (ADPKD) is the most common inheritable form of kidney disease and affects 1:1000 individuals. The root of ADPKD has been tracked to mutations in the PKD1 and PKD2 genes which encode for polycystin subunits. There is no cure for PKD and patients are relegated to survive on dialysis before culminating in kidney transplantation. The hundreds of mutations that lead to PKD are well documented by nephrologists. However, the functional consequences of these variants are poorly characterized compared to the native polycystin function. Primary cilia electrophysiology and structural biology have revealed key aspects of polycystin gating, but these methodologies cannot be rapidly adopted for the characterization of multiple disease variants. The candidate has developed a novel synthetic biology approach for polycystin characterization that can potentially enable faster rates of discovery. The goal of this F32 fellowship proposal is binary. 1) Characterize the functional consequences of ADPKD disease causing variants by implementing a cell-free protein synthesis method for investigating polycystins. The new synthetic biology method will be deployed to characterize a mutation hotspot in PKD2, that relies on post-translational N-glycosylation for its function. In aim 1, PKD2 TOP domain variants will be investigated for membrane integration dynamics and stability of the homotetrameric assembly. In aim 2, the PKD2 variants will be evaluated for their channel function. Lastly, in aim 3, ciliary trafficking of these mutants will be quantified via super resolution imaging. 2) The secondary goal is to provide the applicant with a mentored research and career development training framework to grow as an interdisciplinary scientist in his journey towards a successful independent academic position. To accomplish the training component of this proposal the candidate and his mentoring committee have assembled a training plan tailored to empower his career goals. First, a mentored research training experience in synthetic biology and ion channel biophysics tied to the proposed aims of evaluating the function of PKD2 variants. Second, career development activities to enhance the leadership skills expected of a successful independent investigator. Lastly, scientific training focused in providing hands-on experience in advanced characterization techniques like single particle cryo-EM. By funding this proposal, the candidate will be ideally situated to transition to an independent career and study other dysfunctional human protein targets utilizing innovative biochemical tools.