UC Merced

In order to execute their biological activities, most proteins fold into their unique, three-dimensional structure. The discovery of intrinsically disordered proteins (IDPs) about two decades ago, which are now widely found in eukaryotes, has since challenged the structure-function paradigm. IDPs, which in isolation exist as broad, non-random, conformational ensembles of interconverting states, are centrally involved in many biological processes. The key to their functioning is the ability to fold when bound to ligand partner(s), thus operating as morphing proteins. Despite booming interest in morphing behavior, investigating their structural transitions and mechanism remains extremely difficult because of their distinct characteristics.

Previously, we observed a close connection between intrinsically partially disordered proteins (IPDPS) and gradual (un)folding transitions of downhill folders, leading to the hypothesis that many IPDPs work as a conformational rheostat. The scope of this dissertation is to investigate the biological and technological implications of gradual conformational transitions. We first demonstrate the design principles of protein-based scaffolds by utilizing gradual (un)folding coupled to binding for developing rheostatic conformational transducers using computational modeling and experiments. Our engineered transducers showcase >6 orders of magnitude change in analyte concentration (broadband sensitivity) and have practical advantages over extant ones, which conventionally operate as conformational switches.

Next, inspired by the LEGO toy, we devised a novel modular approach to dissect the folding cooperativity and the energetic contributions of native interactions in defining the conformational ensemble and binding properties of IPDPs. Using an integrated strategy of computation and experiments, we perform an ensemble-based conformational analysis and find that the approach provides an exciting new tool for analyzing morphing transitions that should generally apply to any IPDP, thereby addressing a fundamental gap in the field.

One particularly interesting IPDP is NCBD that binds to multiple structurally diverse ligand partners and recruits the basal transcription machinery. We then explore the concept of NCBD functioning as a conformational rheostat, which allows its promiscuous binding. Finally, using extensive all-atom Molecular Dynamics simulations of NCBD and its biological partners in their free and bound forms, we decipher the hidden conformational biases in the dynamics of the heterogeneous ensemble of NCBD, undergoing gradual morphing transitions hinting at a working conformational rheostat in transcription.