Project Summary The research proposed here seeks to address the structure and dynamics of a PAS regulated protein Kinase (hPASK in humans) using a mix of biochemical and biophysical approaches. The Period-ARNT- Singleminded (PAS) domains, known to sense a wide range of environmental cues to regulate effector proteins. The hPASK maintains an evolutionarily conserved architecture of two PAS domains (PAS-A & PAS- B) and a canonical Serine/Threonine Kinase (STK) domain. The failure to sense and respond to cellular metabolic status and inappropriate lipid accumulation is critical to the pathogenesis of diabetes, obesity, and other metabolic diseases. In both yeast and mammalian cells, hPASK is important in nutrient sensing and metabolic signaling in response to cellular metabolic states. These observations make hPASK an excellent therapeutic candidate for metabolic disease prevention. However, such efforts are limited by incomplete structural and mechanistic characterization of signal transduction within the full length hPASK protein. The sponsor, Dr. Kevin H. Gardner has studied the sensory protein such as PAS domain in both bacterial and human systems, which has led to many significant breakthroughs in therapeutic development. This research plan includes working under the supervision of Dr. Gardner to gain expertise in integrating different structural biology techniques with biochemical data to characterize the structural and functional aspects of this PAS regulated protein kinase. The specific research goals are: 1) to understand the dynamics of the sensory PAS-A domain, and characterize the ligand binding profile within its structure, 2) to determine the Protein- Protein Interactions (PPIs) site between the hPASK domains for its biological function and regulation, and 3) to identify the structural orientation of these multidomain assemblies within the full length protein structure. First, to explore Protein-Ligand Interactions (PLIs) in PAS-A, cutting edge High Pressure (HP) NMR methods, including new techniques pioneered by the Gardner lab and X-ray crystallography, will be used. Second, using biophysical (NMR, FRET, MS) and functional biochemical assays, the molecular mechanism and PPIs sites between the hPASK domain will be identified. Finally, these dynamics and their structural basis between the PAS-A–ligand complex and the PPIs between the PAS and kinase domains will be further characterized within the full length hPASK in the presence and absence of ligand using cryo-EM. Studying the determinants of ligand selectivity within the PAS-A domain and the dynamics of hPASK PLIs and PPIs via biophysical characterization outlined above will enhance researchers understanding of how the signal propagates within the full length hPASK during signaling. Additionally, these biophysical insights will shed light on the potential of hPASK as a therapeutic target in metabolic diseases.