Radical redesign of an allosteric biosensor to respond to new ligands
Abstract
Small molecule ligand biosensors are versatile molecular tools with applications in human health, environmental monitoring, bioremediation and biomanufacturing. Biosensors integrated into the cellular machinery can perform complex biocomputation combining various signals to control cell cycle, cell fate, metabolic output and community behavior. Biosensors are also adept at working in the abiotic environment. For instance, biosensors can interface with electronic, photonic and chemical readouts to make miniature analytical devices that can be deployed in the field. Proteins make excellent small molecule ligand biosensors because of their high specificity, tunable sensitivity, and broad ligand repertoire. Despite the growing importance of biosensors, we lack the capability to design biosensors for new ligands. This is a major limitation impeding progress in utilizing biosensors for diverse applications. We are currently limited to ligands sensed by natural biosensors. Here, we propose to develop a general method to design allosteric biosensors for new ligands using state-of-the-art computational design and high-throughput experimental methods. We will redesign a natural biosensor to respond to diverse ligands radically different from the native ligand. When a small molecule binds to a protein, the protein actuates (or responds) by changing shape, a process known as allostery. Protein residues involved in ligand binding are tightly interconnected with residues involved in allosteric actuation, such that mutating residues to change ligand specificity disrupts allosteric actuation. We will develop high-throughput tools to systematically dissect the role of every residue to understand the molecular rules governing ligand binding and allosteric actuation. We will these principles to develop smarter computational protein design strategies to guide the search toward biosensors that can bind to new ligands while maintaining allosteric actuation. These methods are broadly applicable to design other biosensor protein families such as nuclear receptors, two-component systems and periplasmic binding proteins. With allosteric biosensor QacR as the design candidate, the specific aims are as follows: Aim 1: Building mutation-tolerant scaffolds by stabilizing the wild type biosensor QacR Aim 2: Mapping allosteric hotspots around the ligand-binding pocket by deep mutational scanning. Aim 3: Designing QacR to bind to new ligands radically different from the native ligand.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- May 07, 2018
- Source ID
- W911NF1710043
Entities
People
- Srivatsan Raman
Organizations
- Army Contracting Command
- United States Army
- University of Wisconsin–Madison