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dc.contributor.advisorLoh, Stewart
dc.contributor.authorPresti, Maria
dc.description.abstractProtein-based fluorescent biosensors are powerful tools for analyte recognition in vitro and in cells. Many protein-based binding scaffolds have been developed that recognize ligands with affinity and specificity comparable to those of conventional antibodies, but are smaller, readily overexpressed, and more amenable to engineering. Like antibodies, these binding domains are useful as recognition modules in protein switches and biosensors, but they are not capable of reporting on the binding event by themselves. Here, we engineer two adaptable monobody-based biosensor scaffolds. The first, termed FN3-AFF, is composed of a small binding scaffold-a consensus-designed fibronectin 3 monobody-that it engineered so it undergoes a conformational change upon ligand binding. This change is detected by Förster resonance energy transfer using chemical dyes or cyan and yellow fluorescent proteins as donor/acceptor groups. By grafting substrate recognition residues from different monobodies onto this scaffold, we create fluorescent biosensors for c-Abl Src homology 2 (SH2) domain, WD40-repeat protein 5 (WDR5), small ubiquitin-like modifier-1 (SUMO), and h-Ras. The biosensors bind their cognate ligands reversibly, with affinities consistent with those of the parent monobodies, and with half times of seconds to minutes. We modify the rates of the FN3-AFF switch using a combination of computational strategies and experimental validation to identify rate altering mutations. The resulting mutant results in a 2-fold improvement of kinetics (0.88 s-1 to 1.65 s-1). The second design is an intensiometric biosensor, termed YFP-FN3, created by circularly permuting a naturally occurring fluorescent protein and fusing FN3 of three targets (WDR5, SH2, and HRAS) to it. Ligand binding to the FN3 greatly enhances the fluorescence of the fused fluorescent protein by causing its chromophore to mature. We illustrate the utility of these biosensors for imaging proteins in cells, recognizing endogenous levels of one of the targets, WDR5. These designs serve as generalizable platforms for creating genetically-encoded, ratiometric or intensiometric biosensors by swapping binding residues from known monobodies, with minimal modification.en_US
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 International*
dc.subjectprotein engineeringen_US
dc.subjectalternate frame foldingen_US
dc.titleEngineering modifiable monobody-based biosensor scaffolds for use in vitro and in cellsen_US
dc.description.institutionUpstate Medical Universityen_US
dc.description.departmentBiochemistry & Molecular Biologyen_US
dc.description.degreelevelPhDen_US 2022en_US

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Attribution-NonCommercial-NoDerivatives 4.0 International
Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International