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NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids.

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TLDR
The shortened FAST is designed, which is composed of only 98 amino acids, the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, by truncating 26 N-terminal residues.
Abstract
One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

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Recent Advancements in Tracking Bacterial Effector Protein Translocation

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A unique photochromic UV-A sensor protein, Rc-PYP, interacting with the PYP-binding protein.

TL;DR: In this paper, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated.
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Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore

TL;DR: In this paper , the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen, was described, and two mutants appeared brighter than the wild-type FAST, and these mutants provided up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo.
References
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Journal ArticleDOI

Methods for Measurement of Intermolecular NOEs by Multinuclear NMR Spectroscopy: Application to a Bacteriophage λ N-Peptide/boxB RNA Complex

TL;DR: Improved spectra are obtained relative to data recorded with other purging schemes and structural information obtained for a complex of the amino-terminal arginine rich domain of the N protein from bacteriophage λ and boxB RNA using the new methodology is presented.
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Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.

TL;DR: A platform for rapid generation of highly fluorescent RNA–fluorophore complexes that are optimized for function in cells is described, including a 49-nt aptamer, Broccoli, which shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2.
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The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins.

TL;DR: This review provides both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Journal ArticleDOI

1.4 Å structure of photoactive yellow protein, a cytosolic photoreceptor: Unusual fold, active site, and chromophore

TL;DR: The high-resolution structure of photoactive yellow protein supports a mechanism whereby electrostatic interactions create an active site poised for photon-induced rearrangements and efficient protein-mediated signal transduction.
Journal ArticleDOI

Fluorogen-activating single-chain antibodies for imaging cell surface proteins

TL;DR: The development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens) is reported, isolated by screening a library of human single-chain antibodies using derivatives of thiazole orange and malachite green.
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