Sensing Solvent in the Collapsed State of Glutamine‐Rich Domains

Intrinsically disordered proteins (IDPs) challenge the widely accepted structure to function paradigm of protein behavior and are associated with important biological functions. These disordered domains are estimated to account for at least 30% of the proteome in eukaryotic genomes and are correlated with signal transduction and gene regulation, as well as certain disorders including some types of cancers, amyloidosis, heart disease, and neurodegenerative disease. While many IDPs perform their functions by folding with a partner protein, a subclass is prone to collapsed states, disordered interactions, and aggregation. We need to obtain a higher understanding of the biophysical attributes—level of structures versus collapse and behavior with water in comparison to sequences and biological traits—to characterize these IDPs and to extrapolate their functions. This project takes on two modes: We are (1) developing methods on a model protein known to form a collapsed state that traps water and (2) evaluating a small library of IDPs with distinct roles in transcription by the method developed. Studying these aggregation‐prone disordered domains is challenging. We utilize fluorescence methods to reach low concentrations, an environmentally‐sensitive fluorophore to evaluate the presence of water, and collisional quenching to follow the flow into the interior of the collapsed domain. Our studies of the control protein κ‐casein demonstrate that water is present in the collapsed state but is not able to freely exchange with the bulk solution. Our experimental library of IDPs were selected from auxin response factors (ARFs) from Arabidopsis thaliana . This family of transcription factors have three distinguishable sections: a DNA‐binding domain, a low complexity middle region, and a structured protein:protein interaction domain. My work is centered on the middle region as it is predicted to be disordered across the family but is distinct in abundance of glutamine depending on function. Transcription activators are consistently higher in glutamine than their repressive counterparts. Our final goal is to further investigate this thought by attaching fluorophores to the cysteines of the protein in order to compare the natural and denatured states across our selected proteins. After cloning select sequences from cDNA, we have optimized expression, purification, and fluorophore labeling of four proteins from both transcription activators and repressors. Our comparisons of these will expand our understanding of the sequence‐dependent biophysical traits in the context of their differing biological roles within the context of auxin response in plants.