Journal ArticleDOI
Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity.
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TLDR
Improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins, culminating in a >20-fold increase in contrast.Abstract:
Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to beta-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.read more
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Correlated live cell light and electron microscopy using tetracysteine tags and biarsenicals.
TL;DR: Methods to label cells with biarsenicals, conduct live cell imaging recording sessions, and generate specimens that can be evaluated by EM for a correlated LM/EM analysis are presented.
Journal ArticleDOI
Bimodal Detection of Proteins by 129 Xe NMR and Fluorescence Spectroscopy.
Emilie Mari,Emilie Mari,Yasmina Bousmah,Céline Boutin,Estelle Léonce,Gaëlle Milanole,Thierry Brotin,Patrick Berthault,Marie Erard +8 more
TL;DR: The optimization of a probe for intracellular proteins that combines the advantages of fluorescence and hyperpolarized 129Xe NMR spectroscopy detection is reported.
Journal ArticleDOI
Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCγ1.
Louis Perdios,Tom D. Bunney,Sean C. Warren,Christopher Dunsby,Paul M. W. French,Edward W. Tate,Matilda Katan +6 more
TL;DR: A construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR).
Journal ArticleDOI
Enhanced secretion of recombinant proteins via signal recognition particle (SRP)-dependent secretion pathway by deletion of rrsE in Escherichia coli.
TL;DR: The positive effect of rrsE deficiency on protein secretion via the SRP pathway was successfully demonstrated using various model proteins including endogenous SRP‐dependent proteins, antibodies, and G protein‐coupled receptor.
Journal ArticleDOI
Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries.
TL;DR: The first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries is developed, enabling OBOC libraries to be used in high-throughput discovery of cySteine- rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteINE-rich pharmaceuticals.
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