PROJECT SUMMARY/ABSTRACT Water enables life. So much so that the search for extraterrestrial life is a search for liquid water. However, terrestrial drug discovery still, by and large, ignores water molecules. The reason lies in the complexity and versatility of its contributions to biomolecular interactions, despite its apparent simplicity. My prior research has investigated the role of water in ligand recognition and selectivity, and its utility to improve ligand discovery. Recently, my lab has established a link between the mobility of water networks and the affinity of ligand binding to proteins. More generally, the work highlights the exploitable sensitivity of water networks in response to changes in protein and ligand. My long-term goal is to link atomic-scale water wiggles to changes in the protein conformational landscape and ultimately to organismal fitness. To achieve this, I aspire to pave the path to a more dynamic view of protein function that integrates currently neglected aspects of protein flexibility and hydration. This is important because cryogenic structures, which make up 95% of the Protein Data Bank, deliver a contorted, static image which is then used to design ligands. The objective of the proposed work is to formulate a pragmatic framework that defines and utilizes water network dynamics. As water is ubiquitously found at all biological interfaces, the concept extends beyond protein-ligand interactions to many fields, including protein-protein interactions, allostery, protein evolution and resistance. My central hypothesis is that we can use the exquisite sensitivity of water molecules to contextual changes in our favor. The hypothesis that water fluctuations report on changes in dynamic features will be exploited in three specific areas via perturbation with 1) temperature, 2) mutation and 3) ligands. 1) Re-defining water networks in protein structures: I hypothesize that physiological temperatures provide a less distorted view of water networks within protein crystals than structures obtained using common cryogenic freezing. We will test the hypothesis by solving crystal structures of proteins over a range of temperatures. By exposing freezing artifacts, we can reveal hidden changes in water network dynamics that can be used productively to predict binding affinities. 2) Exploring isoform-specific differences in water networks: I hypothesize that by using water perturbations as a reporter for change, we can distinguish near-identical Hsp90 isoforms. We will test the hypothesis by tracking how co-evolved water networks respond to perturbations in a context-dependent manner. This will expose subtle differences in isoforms that the Hsp90 field has struggled to exploit for decades to explore differential isoform biology in disease. 3) Considering water network dynamics in ligand discovery: I hypothesize that including experimental water network terms will improve computational docking. Based on our previous success of incl...