Non-perturbing and site-specific protein labeling methods are highly desired as they allow researchers to probe complex cellular functions. The biarsenical/tetracysteine labeling system allows in situ fluorescent labeling of intracellular proteins which have been appended with small (12 amino acids) genetically encoded peptide tags. In this work we present the selection of semi-randomized tandem tetracysteine peptides with improved biarsenical (ReAsH) fluorescent brightness (~2-fold) relative to a single tetracysteine motif or rationally designed 3-fold tetracysteine repeat. We found that Fluorescence Activated Cell Sorting by direct ReAsH excitation as opposed to FRET-mediated ReAsH excitation was optimal for selecting 3×Tetracysteine peptides with enhanced brightness. The selected multimer-tetracysteine peptides display enhanced properties due to higher order ReAsH/3×Tetracysteine dye stoichiometries as opposed to enhancement of the individual core tetracysteine photophysical properties. In summary, we have isolated new 3×Tetracysteine motifs with improved ReAsH brightness in live cells. These modular tags should provide enhanced contrast for live cell imaging applications where small tag size (~4.8 KDa) is a requisite for protein labeling.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3031534 | PMC |
| http://dx.doi.org/10.1002/cbic.200900689 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy.
Sci Rep
October 2017
Department of Physics, University of Toronto, Toronto, Ontario, M5S 1A7, Canada.
In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647).
View Article and Find Full Text PDFUsing FlAsH to probe conformational changes in a large HEAT repeat protein.
Chembiochem
May 2012
MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK.
We have investigated the use of FlAsH, a small fluorogenic molecule that binds to tetracysteine motifs, to probe folding of the 15-HEAT repeat protein PR65A. PR65A is one of a special class of modular non-globular proteins known as tandem repeat proteins, which are composed of small structural motifs that stack to form elongated, one-dimensional architectures. We were able to introduce linear and bipartite tetracysteine motifs at several sites along the α-helical HEAT array of PR65A without disrupting the structure or stability.
View Article and Find Full Text PDFSurveying protein structure and function using bis-arsenical small molecules.
Acc Chem Res
September 2011
Department of Chemistry, Yale University, 225 Prospect Street, New Haven, Connecticut 06520-8107, USA.
Exploration across the fields of biology, chemical biology, and medicine has led to an increasingly complex, albeit incomplete, view of the interactions that drive life's processes. The ability to monitor and track the movement, activity, and interactions of biomolecules in living cells is an essential part of this investigation. In our laboratory, we have endeavored to develop tools that are capable not only of monitoring protein localization but also reporting on protein structure and function.
View Article and Find Full Text PDFFACS-based selection of tandem tetracysteine peptides with improved ReAsH brightness in live cells.
Chembiochem
March 2010
Department of Chemistry and Biochemistry, University of Colorado, UCB 215, Boulder, CO 80309, USA.
Non-perturbing and site-specific protein labeling methods are highly desired as they allow researchers to probe complex cellular functions. The biarsenical/tetracysteine labeling system allows in situ fluorescent labeling of intracellular proteins which have been appended with small (12 amino acids) genetically encoded peptide tags. In this work we present the selection of semi-randomized tandem tetracysteine peptides with improved biarsenical (ReAsH) fluorescent brightness (~2-fold) relative to a single tetracysteine motif or rationally designed 3-fold tetracysteine repeat.
View Article and Find Full Text PDFThe monitoring and affinity purification of proteins using dual tags with tetracysteine motifs.
Methods Mol Biol
November 2009
Biosciences Division, Oak Ridge National Laboratory, 37831, Oak Ridge, TN, USA.
Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!