Examination and Reconstruction of Alternate Allosteric Networks in Engineered LacI/GalR Transcription Factors

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This project aims to understand and predict how proteins (i.e., transcription factors) that turn genes on-and-off develop the means to process information and interact with nearby surroundings. The mechanism in which proteins process signal information is called allosteric communication. While the study of allosteric communication has been underway for more than four decades, critical details of these fundamental processes are still poorly understood. This gap in knowledge prevents scientists and engineers from designing new proteins with custom-built communication abilities. Accordingly, the focus of the current project is to elucidate the rules that will enable scientists to build allosteric communication in a family of transcription factors that process a broad range of signals. This research will expand the scientific communities understanding of allosteric communication and enable the development of new tools that can be used to benefit society (e.g., new and less expensive process controls for bio-industrial applications, and systems for use in personalized medicine). Moreover, this project will involve the training and education of the next generation of scientists, specifically Ph.D. level graduate students. Protein allostery is a vitally important protein function that has proven to be a vexing problem to understand at the molecular level. Allosteric communication is a hallmark of many transcription factors used to control gene expression and have enabled synthetic biologist to reprogram cells. The specific goal of this research is to leverage the basic structure and function relationship of the lactose repressor (LacI) to systematically map and engineer alternate allosteric communication, in several LacI/GalR homologues. Answering the fundamental question of whether allostery in the broader LacI/GalR family has been evolved to be controlled by a set of conserved residues or whether the protein is plastic and predisposed to allostery across different residue sequences will ultimately give insights into allosteric structure-function relationships and aid in the engineering of novel transcription factors. Mapping allostery and determining the evolutionary origin, links, and mechanism by which allostery is conferred in the LacI/GalR family will provide crucial knowledge into how other proteins may be designed to exhibit allostery and how to control and modulate allostery in existing systems. Upon completion, this study will enable scientist to test assertions with regard to the origin and molecular mechanics of alternate allosteric communication via the development of design rules for specific allosteric operations. In turn this algorithm will produce novel LacI/GalR-based transcription factors for use in a broad range of biotechnological applications. This project is co-funded by the Molecular Biophysics and the Systems and Synthetic Biology programs of the Division of Molecular and Cellular Biosciences.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.