Project Summary: The confinement, localization, and nanoscale organization of biomolecules can dramatically alter their biochemical and physiological properties creating opportunities for in vivo sensing and enhancing their use as therapeutics. While these principles are relatively well-studied for oligonucleotides, which are frequently used as vaccines, antisense therapeutics, and theranostics, they are less well-studied for short biological peptides of similar lengths (10-100s of amino acids). One particularly relevant class of peptides are intrinsically disordered proteins (IDPs), which are highly reconfigurable in response to environmental stimuli, including temperature, pH, ionic strength, and mechanical force. While fundamental studies have revealed some of these effects on solution peptides, understanding these properties for IDPs that are confined or organized at the nanoscale is less well understood. This represents a significant knowledge gap because understanding and controlling the dynamic properties of IDPs at the nanoscale could enable the creation of stimuli-responsive in vivo pH sensors while providing insights into the impact of confinement on clinically relevant IDPs regions. The long- term goal of our work is to develop a comprehensive and forward-looking framework for understanding, and ultimately designing, the biophysical properties of IDP-nanoparticle architecture. The objective of this project is to reveal the sequence-, density-, and stimuli-dependence of IDPs bound to small (~10 nm) spherical nanoparticles, and its ability to influence and control the physical properties of nanoparticle core. The central hypothesis of this work, based upon previous work on DNA-nanoparticle conjugates (Ross) and IDP pH sensitivity (Gage), is that localization and confinement of IDPs onto nanoparticle constructs will influence their structural plasticity to pH, temperature, and ionic strength. Consequently, IDP-nanoparticle conjugates can generated with tunable responsivity to their environment, leveraging the biophysical properties of the IDPs and the physical properties of the inorganic nanoparticle core. The approach for testing our central hypothesis comprises two specific aims: 1) Reveal the distinct biophysical properties of nanoparticle-bound IDPs compared to free-in-solution IDPs; and 2) Determine how IDP sequence and density influences nanoscale optical readouts for sensing ionic strength, temperature, and pH changes. This study is innovative as it represents one of the first approaches to coupling IDPs to nanoparticles and the insights gained from this study are necessary to develop a system that is tunable for specific environmental changes. These studies are a critical first step towards developing new IDP- nanoparticle sensors that will have a range of biomedical applications.