Dataset Information

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process.

ABSTRACT: Many biotechniques use complementary split-fluorescent protein (sFPs) fragments to visualize protein-protein interactions, image cells by ensemble or single molecule fluorescence microscopy, or assemble nanomaterials and protein superstructures. Yet, the reassembly mechanisms of sFPs, including fragment binding rates, folding, chromophore maturation and overall photophysics remain poorly characterized. Here, we evolved asymmetric and self-complementing green, yellow and cyan sFPs together with their full-length equivalents (flFPs) and described their biochemical and photophysical properties in vitro and in cells. While re-assembled sFPs have spectral properties similar to flFPs, they display slightly reduced quantum yields and fluorescence lifetimes due to a less sturdy ?-barrel structure. The complementation of recombinant sFPs expressed in vitro follows a conformational selection mechanism whereby the larger sFP fragments exist in a monomer-dimer equilibrium and only monomers are competent for fluorescence complementation. This bimolecular fragment interaction involves a slow and irreversible binding step, followed by chromophore maturation at a rate similar to that of flFPs. When expressed as fusion tags in cells, sFPs behave as monomers directly activated with synthetic complementary fragments. This study resulted in the development of sFP color variants having improved maturation kinetics, brightness, and photophysics for fluorescence microscopy imaging of cellular processes, including single molecule detection.

SUBMITTER: Koker T

PROVIDER: S-EPMC5871787 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Publications

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process.

Köker Tuğba T Fernandez Anthony A Pinaud Fabien F

Scientific reports 20180328 1

Many biotechniques use complementary split-fluorescent protein (sFPs) fragments to visualize protein-protein interactions, image cells by ensemble or single molecule fluorescence microscopy, or assemble nanomaterials and protein superstructures. Yet, the reassembly mechanisms of sFPs, including fragment binding rates, folding, chromophore maturation and overall photophysics remain poorly characterized. Here, we evolved asymmetric and self-complementing green, yellow and cyan sFPs together with t ...[more]

PMID: 29593344

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Project description:Listeria monocytogenes is an intracellular food-borne pathogen that has evolved to enter mammalian host cells, survive within them, spread from cell to cell, and disseminate throughout the body. A series of secreted virulence proteins from Listeria are responsible for manipulation of host-cell defense mechanisms and adaptation to the intracellular lifestyle. Identifying when and where these virulence proteins are located in live cells over the course of Listeria infection can provide valuable information on the roles these proteins play in defining the host-pathogen interface. These dynamics and protein levels may vary from cell to cell, as bacterial infection is a heterogeneous process both temporally and spatially. No assay to visualize virulence proteins over time in infection with Listeria or other Gram-positive bacteria has been developed. Therefore, we adapted a live, long-term tagging system to visualize a model Listeria protein by fluorescence microscopy on a single-cell level in infection. This system leverages split-fluorescent proteins, in which the last strand of a fluorescent protein (a 16-amino-acid peptide) is genetically fused to the virulence protein of interest. The remainder of the fluorescent protein is produced in the mammalian host cell. Both individual components are nonfluorescent and will bind together and reconstitute fluorescence upon virulence-protein secretion into the host cell. We demonstrate accumulation and distribution within the host cell of the model virulence protein InlC in infection over time. A modular expression platform for InlC visualization was developed. We visualized InlC by tagging it with red and green split-fluorescent proteins and compared usage of a strong constitutive promoter versus the endogenous promoter for InlC production. This split-fluorescent protein approach is versatile and may be used to investigate other Listeria virulence proteins for unique mechanistic insights in infection progression.

Dataset Information

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process.

Publications

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process.

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