Titlebook: Synthetic Protein Switches; Methods and Protocol Viktor Stein Book 2017 Springer Science+Business Media LLC 2017 Bioluminescent sensors.Flu

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Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Raoral resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FR

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Method for Developing Optical Sensors Using a Synthetic Dye-Fluorescent Protein FRET Pair and Computoviding detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their d

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Rational Design and Applications of Semisynthetic Modular Biosensors: SNIFITs and LUCIDse outline the design of two types of modular biosensors: .AP-tag-based .ndicators with a .luorescent .ntramolecular .ether (SNIFITs) and .iferase-based .ndicators of .rugs (LUCIDs). These semisynthetic biosensors quantitatively measure analyte concentrations in vitro and on cell surfaces by an intra

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Ultrasensitive Firefly Luminescent Intermediate-Based Protein-Protein Interaction Assay (FlimPIA) Ba the catalytic mechanism of firefly luciferase (Fluc) that can be divided into two half-reactions: the adenylation step and the oxidative luminescent steps. We engineered two mutant Fluc enzymes named “Donor” and “Acceptor” where the oxidative luminescent activity of the Donor is almost eliminated a

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Quantitative and Dynamic Imaging of ATM Kinase Activitys). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays

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Creation of Antigen-Dependent β-Lactamase Fusion Protein Tethered by Circularly Permuted Antibody Vaulating the catalytic activity of enzymes by antibodies is still hard to achieve. Here, we describe a design method of unique antibody variable region Fv introduced with two circular permutations, called Clampbody. By tethering the Clampbody to a circularly permuted TEM-1 β-lactamase (BLA), we succe

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Protein and Protease Sensing by Allosteric Derepressionn of these events is therefore indispensable for a detailed understanding of cellular regulation. Here, we present versatile and modular sensors that allow the user to detect protease activity and protein-peptide interactions, as well as to screen for inhibitors using chromogenic, fluorescent, or lu

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DNA-Specific Biosensors Based on Intramolecular β-Lactamase-Inhibitor Complex Formationucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-β-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzy

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Characterizing Dynamic Protein–Protein Interactions Using the Genetically Encoded Split Biosensor Asanding of signaling in health and disease. Genetically encoded split protein biosensor assays, such as the split TEV method, have proved to be highly valuable when studying regulated PPIs in living cells. Split TEV is based on the functional complementation of two previously inactive TEV protease fr