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

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• 作者：Aurlien Dif ; Fouzia Boulmedais ; Mathieu Pinot ; Victor Roullier ; Michle Baudy-Floc’ ; h ; Frdric M. Coquelle ; Samuel Clarke ; Pierre Neveu ; Franoise Vignaux ; Roland Le Borgne ; Maxime Dahan ; Zoher Guer
• 刊名：Journal of the American Chemical Society
• 出版年：2009
• 出版时间：October 21, 2009
• 年：2009
• 卷：131
• 期：41
• 页码：14738-14746
• 全文大小：355K
• 年卷期：v.131,no.41(October 21, 2009)
• ISSN：1520-5126

文摘

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.