Small and stable peptidic PEGylated quantum dots to target polyhistidine-tagged proteins with controlled stoichiometry.
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.
Université de Rennes 1, CNRS UMR 6226, Sciences Chimiques de Rennes, Rennes, France.
Aurélien
A
Dif
Fouzia
F
Boulmedais
Mathieu
M
Pinot
Victor
V
Roullier
Michèle
M
Baudy-Floc'h
Frédéric M
FM
Coquelle
Samuel
S
Clarke
Pierre
P
Neveu
Françoise
F
Vignaux
Roland
R
Le Borgne
Maxime
M
Dahan
Zoher
Z
Gueroui
Valérie
V
Marchi-Artzner
D000818:Animals; D002470:Cell Survival; D005110:Extracellular Space / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008027:Light; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D009068:Movement; D010455:Peptides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability; D021381:Protein Transport; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D045663:Quantum Dots; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D008941:Spindle Apparatus / Q000378:metabolism; D013194:Staining and Labeling; D013379:Substrate Specificity
C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D015335:Molecular Probes; D010455:Peptides; D011506:Proteins; C033223:polyhistidine; D011092:Polyethylene Glycols; D006639:Histidine