CAREER: Defining structural principles for the engineering and evolution of allostery

About This Grant

Proteins are nanometer-scale molecules whose composition and shape are specified by genes. Inside the cell, they catalyze chemical reactions, perform mechanical work, and assemble into larger structures. The collective action of very many proteins drives cell growth, division, and movement. Proteins are often controlled by external signals – for example, adding a nutrient to the cellular environment might turn a protein “on” or “off” by binding to the protein and altering its shape or dynamics. Such allosteric control provides a basic mechanism for cells to sense and respond to the environment and is a building block for intracellular communication. A complete understanding of how allosteric control works, and how it is encoded in the genetic sequence of proteins, would allow biologists to engineer proteins that respond to artificial cues. In this project, the PI’s research team will use computation and experiment to understand how allosteric regulation in a protein is optimized, define physical properties distinguishing allosteric surfaces, and construct a set of synthetic allosteric switches that enable control of cell growth rate with light. This work will establish a practical, general toolkit for engineering allosteric regulation, and provide fundamental insights into how natural allosteric regulation might evolve. This proposal will train graduate, undergraduate, and bio-oriented high school students to improve their skills in basic programming and research experimentation to contribute STEM workforce development. Though allostery is a fundamental and common feature of proteins, how it is encoded by protein sequence and structure remains unclear. For example, it is unknown if the pattern and number of mutations influencing allostery is sparse or abundant, or if the mutations with the biggest effect on regulation are localized to the allosteric site or distributed throughout the structure. This project will address these fundamental questions by using deep mutational scanning to characterize the complete set of mutations than can influence allosteric regulation in a synthetic allosteric switch. This research will also use new approaches in NMR spectroscopy to better understand how protein interactions with water might determine (and identify) allosteric surfaces. The hypothesis is that allosteric surfaces are marked by slowed hydration dynamics. If so, this would suggest that allosteric sites are entropically preferred sites for the evolution of new protein and ligand binding interactions. Moreover, this would open new avenues for the discovery of allosterically acting pharmaceuticals. Finally, the knowledge gained through these mutational and structural studies will be applied to generate a series of broad dynamic range allosteric switches for the light-based control of cell growth. These switches will provide a practical toolkit for dynamically modulating cell growth, with potential applications in the study of bacterial community dynamics, eukaryotic cell proliferation, and engineering live biotherapeutics. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.