Journal ArticleDOI
Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells
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TLDR
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.Abstract:
Recombinant proteins containing four cysteines at the i , i + 1, i + 4, and i + 5 positions of an α helix were fluorescently labeled in living cells by extracellular administration of 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein. This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility in attachment sites as well as potential spectroscopic and chemical properties. 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.read more
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Book ChapterDOI
Probing the plasma membrane organization in living cells by spot variation fluorescence correlation spectroscopy.
Cyrille Billaudeau,Sébastien Mailfert,Sébastien Mailfert,Sébastien Mailfert,Tomasz Trombik,Tomasz Trombik,Tomasz Trombik,Nicolas Bertaux,Nicolas Bertaux,Vincent Rouger,Vincent Rouger,Vincent Rouger,Yannick Hamon,Yannick Hamon,Yannick Hamon,Hai-Tao He,Hai-Tao He,Hai-Tao He,Didier Marguet,Didier Marguet,Didier Marguet +20 more
TL;DR: A method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization is described, based on questioning the relationship between the diffusion time τ(d) and the squared waist of observation w(2).
Journal ArticleDOI
A designed protein for the specific and covalent heteroconjugation of biomolecules.
TL;DR: This work introduces the molecular adaptor covalin, which is a protein chimera of two self-labeling proteins with nonoverlapping substrate specificity that permits a selective and covalent heteroconjugation of biomolecules displaying appropriate functional groups.
Journal ArticleDOI
The molecular mechanism of heme loss from oxidized soluble guanylate cyclase induced by conformational change
Jie Pan,Xiaoxue Zhang,Hong Yuan,Qiming Xu,Huijuan Zhang,Yajun Zhou,Zhong-Xian Huang,Xiangshi Tan +7 more
TL;DR: It is proposed that the synergistic effect of these conformational changes of the discrete region upon heme oxidation forces the heme pocket open, and subsequent heme loss readily.
Journal ArticleDOI
Breeding and building molecules to spy on cells and tumors
TL;DR: Genetically targeted labels have been demonstrated for many important signals, including membrane potential, intracellular calcium, cyclic nucleotides, and redox status at least at the level of thiols and disulfides, although maybe not quite the same electron transport chain as the authors just heard about from Dr Fujibayashi.
Journal ArticleDOI
Engineered kinesin motor proteins amenable to small-molecule inhibition.
Martin F. Engelke,Michael Winding,Yang Yue,Shankar Shastry,Shankar Shastry,Federico Teloni,Sanjay Reddy,T. Lynne Blasius,Pushpanjali Soppina,William O. Hancock,Vladimir I. Gelfand,Kristen J. Verhey +11 more
TL;DR: A novel chemical-genetic approach is presented to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules.
References
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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.
Journal ArticleDOI
Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.
François Denizot,Rita Lang +1 more
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Journal ArticleDOI
Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin
Atsushi Miyawaki,Juan Llopis,Roger Heim,J. Michael McCaffery,Joseph A. Adams,Mitsuhiko Ikura,Mitsuhiko Ikura,Roger Y. Tsien +7 more
TL;DR: New fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations are constructed and dubbed ‘cameleons’.
Journal ArticleDOI
Crystal structure of the Aequorea victoria green fluorescent protein.
TL;DR: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products.
Journal ArticleDOI
Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer
Roger Heim,Roger Y. Tsien +1 more
TL;DR: The results demonstrate that the production of more and better GFP variants is possible and worthwhile, and facilitates multicolor imaging of differential gene expression, protein localization or cell fate.
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