Journal ArticleDOI
New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.
Stephen R. Adams,Robert E. Campbell,Larry A. Gross,Brent R. Martin,Grant K. Walkup,Yong Yao,Juan Llopis,Roger Y. Tsien +7 more
TLDR
Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix.Abstract:
We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269−272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565−578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants (∼10-11 M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed α-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable a...read more
Citations
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Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics
Xavier Michalet,Fabien Pinaud,Laurent A. Bentolila,James M. Tsay,Sören Doose,J. Jack Li,Gobalakrishnan Sundaresan,Anna M. Wu,Sanjiv S. Gambhir,Sanjiv S. Gambhir,Shimon Weiss +10 more
TL;DR: The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.
Journal ArticleDOI
The fluorescent toolbox for assessing protein location and function
TL;DR: The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy.
Journal ArticleDOI
Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality
TL;DR: The bioorthogonal chemical reactions developed to date are described and how they can be used to study biomolecules.
Journal ArticleDOI
Engineering and characterization of a superfolder green fluorescent protein.
Jean-Denis Pedelacq,Stéphanie Cabantous,Timothy H. Tran,Thomas C. Terwilliger,Geoffrey S. Waldo +4 more
TL;DR: A robustly folded version of GFP is generated, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides, and shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics.
Journal ArticleDOI
Creating new fluorescent probes for cell biology.
TL;DR: Advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein–protein interactions.
References
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Multicolor and Electron Microscopic Imaging of Connexin Trafficking
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Immobilized metal ion affinity chromatography
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