De novo design of allosterically regulated proteins to study intramolecular signal transduction

About This Grant

This proposal aims to design proteins from scratch that can undergo intramolecular allosteric regulation, a process crucial to central metabolism, genetic regulatory networks, and biological sensing. If we truly understand the molecular biomechanics behind allosteric regulation, then we should be able to design proteins that use remote binding of a ligand to control small molecule release. Recent work has demonstrated the possibility of changing remote protein conformation through fold switching or domain displacement. Researchers from my postdoctoral training lab have shown that designed proteins can bind small molecules with sub-nanomolar affinity, designed enzymes can use a remote binding event to suppresses reactivity, and designed membrane proteins acting as part of a protein complex can transmit information from one binding event to influence distant kinase activity. Building from these recent advances in protein design, as well as the wealth of experimental data on signal transduction in helical bundle proteins such as G-protein coupled receptors and histidine kinases, the proposed research aims to develop new helical bundle proteins where binding of a ligand at one site increases the population of protein in a strained conformation, causing small molecule release at a second site. The capacity to design targeted proteins capable of small molecule delivery in response to changes in ligand concentration would give rise to powerful new capacities for biotechnology, and this proposal aims to explore release of two drugs for which de novo protein binders have been recently designed. In Aim 1, I will design a protein that releases the anti-cancer drug rucaparib upon binding of the pro-tumor metabolite itaconate. In Aim 2, I will design a protein that releases the anti-cancer drug apixaban following itaconate binding. Binding sites for the allosteric ligand and releasable molecule will be incorporated onto protein scaffolds built from stable helical bundles in combination with loops and kinks generated from diffusion-based probabilistic models. The resulting proteins will be expressed and purified, their binding will be assayed using fluorescence spectroscopy, and their structures will be determined using X-ray crystallography and NMR spectroscopy. Once successful allosterically regulated proteins are designed, new ligand binding sites will be positioned increasingly distal to the active site to explore progressively more remote strain transduction. Demonstrating intramolecular allosteric regulation within a single protein monomer will extend our understanding of a longstanding problem in protein biophysics. The proposed research project will advance my training goals as I integrate computational protein design, molecular biology and biochemistry techniques for cloning, protein expression and purification, and biophysical characterization. Combining my existing experience in physical chemistry with the skills developed during the proposed research will prepare me for an independent research career using proteins and synthetic macromolecules to understand and intervene in strained biological macromolecules.