Journal ArticleDOI
Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity.
Reads0
Chats0
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
Citations
More filters
Journal ArticleDOI
ReAsH as a Quantitative Probe of In-Cell Protein Dynamics.
TL;DR: Fast relaxation imaging (FReI), combining millisecond temperature jump kinetics with fluorescence microscopy detection, circumvents many of the difficulties encountered working with the ReAsH system, allowing us to obtain quantitative FRET measurements of protein stability and kinetics both in vitro and in cells.
Journal ArticleDOI
Arf6 controls beta-amyloid production by regulating macropinocytosis of the Amyloid Precursor Protein to lysosomes
Weihao Tang,Weihao Tang,Joshua H. K. Tam,Joshua H. K. Tam,Claudia Seah,Justin Chiu,Justin Chiu,Andrea Tyrer,Andrea Tyrer,Sean P. Cregan,Sean P. Cregan,Susan O. Meakin,Susan O. Meakin,Stephen H. Pasternak,Stephen H. Pasternak +14 more
TL;DR: It is found that APP can be rapidly internalized from the cell surface to lysosomes, bypassing early and late endosomes and suggesting that altering APP trafficking represents a viable strategy to reduce Aβ production.
Journal ArticleDOI
A FlAsH‐Based Cross‐Linker to Study Protein Interactions in Living Cells
TL;DR: A dimeric derivative of the arsenical compound FlAsH enables the highly specific, covalent cross-linking of two proteins containing a 12 amino acid peptide tag, which can be used to detect and manipulate protein-protein interactions both in vitro and in living cells.
Journal ArticleDOI
Protein tango: the toolbox to capture interacting partners.
Anna Rutkowska,Carsten Schultz +1 more
TL;DR: Which cross-linkers and inducers of dimerization are out there and how to make use of this great toolbox are described.
Journal ArticleDOI
Exploration of Biarsenical Chemistry—Challenges in Protein Research
Adam Pomorski,Artur Krężel +1 more
TL;DR: This work focuses on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo, and has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein–protein interaction studies, protein stability, and aggregation studies.
References
More filters
Journal ArticleDOI
The green fluorescent protein
TL;DR: In just three years, the green fluorescent protein from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology.
PatentDOI
FACS-optimized mutants of the green fluorescent protein (GFP)
TL;DR: In this article, three classes of GFP mutants having single excitation maxima around 488 nm are shown to be brighter than wild-type GFP following 488-nm excitation.
PatentDOI
Green fluorescent protein
Martin Chalfie,Douglas Prasher +1 more
TL;DR: This demonstration indicated that GFP could be used as a marker of gene expression and protein localization in living and fixed tissues and variations with more intense fluorescence or alterations in the excitation and emission spectra have been produced.
Journal ArticleDOI
Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells
TL;DR: This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.
Journal ArticleDOI
An improved cyan fluorescent protein variant useful for FRET.
TL;DR: Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.