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Small and stable peptidic PEGylated quantum dots to target polyhistidine-tagged proteins with controlled stoichiometry.

Abstract : The use of the semiconductor quantum dots (QD) as biolabels for both ensemble and single-molecule tracking requires the development of simple and versatile methods to target individual proteins in a controlled manner, ideally in living cells. To address this challenge, we have prepared small and stable QDs (QD-ND) using a surface coating based on a peptide sequence containing a tricysteine, poly(ethylene glycol) (PEG), and an aspartic acid ligand. These QDs, with a hydrodynamic diameter of 9 +/- 1.5 nm, can selectively bind to polyhistidine-tagged (histag) proteins in vitro or in living cells. We show that the small and monodisperse size of QD-ND allows for the formation of QD-ND/histag protein complexes of well-defined stoichiometry and that the 1:1 QD/protein complex can be isolated and purified by gel electrophoresis without any destabilization in the nanomolar concentration range. We also demonstrate that QD-ND can be used to specifically label a membrane receptor with an extracellular histag expressed in living HeLa cells. Here, cytotoxicity tests reveal that cell viability remains high under the conditions required for cellular labeling with QD-ND. Finally, we apply QD-ND complexed with histag end binding protein-1 (EB1), a microtubule associated protein, to single-molecule tracking in Xenopus extracts. Specific colocalization of QD-ND/EB1 with microtubules during the mitotic spindle formation demonstrates that QD-ND and our labeling strategy provide an efficient approach to monitor the dynamic behavior of proteins involved in complex biological functions.
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https://www.hal.inserm.fr/inserm-00422050
Contributor : Hervé de Villemeur <>
Submitted on : Wednesday, September 21, 2011 - 2:58:01 PM
Last modification on : Monday, November 23, 2020 - 10:30:09 AM
Long-term archiving on: : Thursday, December 22, 2011 - 2:20:31 AM

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Aurélien Dif, Fouzia Boulmedais, Mathieu Pinot, Victor Roullier, Michèle Baudy-Floc'H, et al.. Small and stable peptidic PEGylated quantum dots to target polyhistidine-tagged proteins with controlled stoichiometry.. Journal of the American Chemical Society, American Chemical Society, 2009, 131 (41), pp.14738-46. ⟨10.1021/ja902743u⟩. ⟨inserm-00422050⟩

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