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.
Dif
Aurélien
A
Université de Rennes 1, CNRS UMR 6226, Sciences Chimiques de Rennes, Rennes, France.
Boulmedais
Fouzia
F
Pinot
Mathieu
M
Roullier
Victor
V
Baudy-Floc'h
Michèle
M
Coquelle
Frédéric M
FM
Clarke
Samuel
S
Neveu
Pierre
P
Vignaux
Françoise
F
Le Borgne
Roland
R
Dahan
Maxime
M
Gueroui
Zoher
Z
Marchi-Artzner
Valérie
V
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