Project Summary/Abstract: The emergence of unprecedented high-resolution structures of biological building blocks is revolutionizing the way we can rationalize the protein machinery, yet the structure only offers a necessary, but insufficient, starting point to uncover protein property, activity and function. There is a fundamental knowledge gap in understanding the translation of protein structure and surface chemistry into protein property and function. Even a property as widely studied as protein solubility cannot be a priori predicted from a known protein structure with our current tools and knowledge, as underscored by the observation that many single mutations invoke minimal structural changes while dramatically changing the stability, property and aggregability. Our vision is to uncover the code for translating protein surface structural properties into protein surface activity, interaction and function. The Han lab is working on achieving such translation, among others, aided by advanced spectroscopy methods that probe local protein dynamics, site-specific hydration properties and conformational ensembles. These measurements are enabled by existing state-of-the-art tools, such as electron paramagnetic resonance (EPR) lineshape analysis, pulsed dipolar EPR and solid-state nuclear magnetic resonance (NMR), as well as novel approaches developed by the Han lab, such as Overhauser dynamic nuclear polarization (ODNP) and other DNP-amplified NMR methods. The combination of these methods will enable the detection of protein surface hydration, topology and interaction, in dilute solution state under physiological conditions and with enhanced sensitivity. The Han lab will systematically apply and refine the tools and methods to study protein stability, interaction, phase separation, oligomerization to aggregation. The five-year goals focus on the following select classes of proteins. We choose the globular protein LOV as a model to uncover the structure-function relationship to aid in rational protein engineering leading to properties such as enhanced binding affinity, allostery, fluorescent property and controlled conformational plasticity. We choose the protein tau to study an intrinsically disordered protein (IDP) for which single-point mutations and subtle partner interactions significantly tune its stability and aggregation propensity in disease contexts. Our goal will be to reveal the mechanisms, e.g. by hydration perturbation or conformational ensemble shifts, through which mutations and other modifications modulate aggregation propensity. Finally, we aim to uncover the structural and mechanistic basis, as well as functional consequences, of oligomerization of two trans-membrane proteins, PR and A2A. The long-term goal of this proposal is to decipher the design rules for interactions and active surfaces of proteins, so that from the protein surface structure and topology one can predict where the binding site is, or learn how to design one that rationally modulates protein-protein assembly, and select or design specific aggregation pathways.