Caged Activators of Artificial Allosteric Protein Biosensors
journal contributionposted on 14.05.2020, 13:09 by Selvakumar Edwardraja, Zhong Guo, Jason Whitfield, Ignacio Retamal Lantadilla, Wayne A. Johnston, Patricia Walden, Claudia E. Vickers, Kirill Alexandrov
The ability of proteins to interconvert unrelated biochemical inputs and outputs underlays most energy and information processing in biology. A common conversion mechanism involves a conformational change of a protein receptor in response to a ligand binding or a covalent modification, leading to allosteric activity modulation of the effector domain. Designing such systems rationally is a central goal of synthetic biology and protein engineering. A two-component sensory system based on the scaffolding of modules in the presence of an analyte is one of the most generalizable biosensor architectures. An inherent problem of such systems is dependence of the response on the absolute and relative concentrations of the components. Here we use the example of two-component sensory systems based on calmodulin-operated synthetic switches to analyze and address this issue. We constructed “caged” versions of the activating domain thereby creating a thermodynamic barrier for spontaneous activation of the system. We demonstrate that the caged biosensor architectures could operate at concentrations spanning 3 orders of magnitude and are applicable to electrochemical, luminescent, and fluorescent two-component biosensors. We analyzed the activation kinetics of the caged biosensors and determined that the core allosteric switch is likely to be the rate limiting component of the system. These findings provide guidance for predictable engineering of robust sensory systems with inputs and outputs of choice.
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effector domain3 ordersallosteric activity modulationCaged Activatorsbiosensor architecturesprotein receptoroutputs underlaysactivation kineticscore allosteric switchprotein engineeringgeneralizable biosensor architecturesligand bindingcovalent modificationconversion mechanisminformation processingArtificial Allosteric Protein Biosensors