Small and Stable Peptidic PEGylated Quantum Dots to Target Polyhistidine-Tagged Proteins with Controlled Stoichiometry

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TitreSmall and Stable Peptidic PEGylated Quantum Dots to Target Polyhistidine-Tagged Proteins with Controlled Stoichiometry
Type de publicationJournal Article
Nouvelles publications2009
AuteursDif, A, Boulmedais F, Pinot M, Roullier V, Baudy-Floc'h M, Coquelle FM, Clarke S, Neveu P, Vignaux F, Le Borgne R, Dahan M, Gueroui Z, Marchi-Artzner V
JournalJournal of the American Chemical Society
Volume131
Fascicule41
Pagination14738-14746
Année de publicationOct
Numéro0002-7863
Accession NumberISI:000271271800045
Résumé

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 OD-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.

URL<Go to ISI>://000271271800045
DOI10.1021/ja902743u
Importer un fichierSmall and Stable Peptidic PEGylated Quantum Dots to Target Polyhistidine-Tagged Proteins with Controlled Stoichiometry
Unité de rattachement: 
UMR 8640