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A general strategy to red-shift green fluorescent protein-based biosensors.

ABSTRACT: Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic ?-cells.

SUBMITTER: Zhang S

PROVIDER: S-EPMC7669575 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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A general strategy to red-shift green fluorescent protein-based biosensors.

Zhang Shen S Ai Hui-Wang HW

Nature chemical biology 20200914 12

Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we des ...[more]

PMID: 32929278

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Project description:Leave-one-out green fluorescent protein (LOOn-GFP) is a circularly permuted and truncated GFP lacking the nth ?-strand element. LOO7-GFP derived from the wild-type sequence (LOO7-WT) folds and reconstitutes fluorescence upon addition of ?-strand 7 (S7) as an exogenous peptide. Computational protein design may be used to modify the sequence of LOO7-GFP to fit a different peptide sequence, while retaining the reconstitution activity. Here we present a computationally designed leave-one-out GFP in which wild-type strand 7 has been replaced by a 12-residue peptide (HA) from the H5 antigenic region of the Thailand strain of H5N1 influenza virus hemagglutinin. The DEEdesign software was used to generate a sequence library with mutations at 13 positions around the peptide, coding for approximately 3 × 10(5) sequence combinations. The library was coexpressed with the HA peptide in E. coli and colonies were screened for in vivo fluorescence. Glowing colonies were sequenced, and one (LOO7-HA4) with 7 mutations was purified and characterized. LOO7-HA4 folds, fluoresces in vivo and in vitro, and binds HA. However, binding results in a decrease in fluorescence instead of the expected increase, caused by the peptide-induced dissociation of a novel, glowing oligomeric complex instead of the reconstitution of the native structure. Efforts to improve binding and recover reconstitution using in vitro evolution produced colonies that glowed brighter and matured faster. Two of these were characterized. One lost all affinity for the HA peptide but glowed more brightly in the unbound oligomeric state. The other increased in affinity to the HA peptide but still did not reconstitute the fully folded state. Despite failing to fold completely, peptide binding by computational design was observed and was improved by directed evolution. The ratio of HA to S7 binding increased from 0.0 for the wild-type sequence (no binding) to 0.01 after computational design (weak binding) and to 0.48 (comparable binding) after in vitro evolution. The novel oligomeric state is composed of an open barrel.

Dataset Information

A general strategy to red-shift green fluorescent protein-based biosensors.

Publications

A general strategy to red-shift green fluorescent protein-based biosensors.

Similar Datasets

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