Quantum Dots to Target Polyhistidine-Tagged Proteins
A R T I C L E S
gies have now been described to obtain bioconjugated QD
probes, including both covalent or noncovalent interactions of
proteins or other biomolecules at the QD surface. These include
the DHLA strategy, QD/protein complexes exhibit a reduced
size (hydrodynamic diameter ∼15 nm) in comparison to QDs
32
2
9-31
with amphiphilic molecule (∼20-30 nm)
or polymer-based
7
,11,12
14
specific recognition system such as streptavidin/biotin,
(∼20-30 nm) surface coatings.
1
3
antibody-antigen recognition, or electrostatic interactions.
In the present work, we report a simple alternative for labeling
individual histag proteins with QDs while maintaining a small
QD/protein complex size. We selected short peptides to stabilize
the QDs in aqueous buffers because of their good chemical
General concerns in all these methods are the final size of the
1
5,16
bioconjugate,
the multivalency of the conjugation, and the
1
7
preservation of protein functionality. Since the size of a QD
is comparable or greater than the size of many proteins, it is
technically difficult to separate QD/protein complexes with
different stoichiometries. Only recently, it was reported that gel
electrophoresis can be used to isolate QD/streptavidin/antibody
versatility as well as their higher chemical stability compared
33,34
with QD coatings formed with ester bonds.
Peptides also
have the advantage that it is easy to introduce biologically active
sequences as terminal groups and they can be obtained from
commercial providers, eliminating the need for complex or time-
consuming chemical synthesis and characterization. Peptides
derived from the phytochelatin were first demonstrated to be a
very effective surface coating, yielding water-soluble QDs with
1
8
complexes with 1:1 stoichiometry.
To target a specific site on a protein of interest, one strategy
1
9
is to introduce a small fusion tag such as a polyhistidine
peptide sequence (histag). This tag, commonly used for purifica-
tion, contains six (H6) or ten histidines (H10) and exhibits a
35
hydrodynamic diameters of ∼10-15 nm. We have found that
2
+
2+
high affinity for divalent nickel (Ni ) or zinc (Zn ) ions.
Previously, we reported the preparation of QD-micelles bearing
a tris-nitrilotriacetic acid (trisNTA) ligand to target histag
small tricysteine peptide derivatives, such as the sequence
CCCSSSD, can also interact strongly with the QD surface and
serve to form stable water-soluble QDs with a hydrodynamic
2+
20
proteins by high-affinity complexation via Ni ions. However,
a possible limitation of these QD-micelles is that their hydro-
dynamic diameter is between 20 to 30 nm following complex-
ation with the histag protein. Since intracellular trafficking of
36
diameter of ∼10 nm. On the basis of these results, we prepared
small QDs based on peptide ND, composed of a tricysteine
adhesive group, a hexamer of PEG, and an aspartic acid grafted
to the terminal group. We show that for these QD-ND, the PEG
spacer effectively prevents nonspecific adsorption on cell
membranes and that the probe is nontoxic to cells under standard
experimental conditions. Furthermore, specific binding of histag
proteins is mediated by binding to Zn2 ions directly accessible
on the QD surface and possibly enhanced by the metal-chelating
dicarboxylate acid functionality of the aspartic acid.
2
1
nanoparticles and their access to crowded cellular environ-
2
2
ments are strongly influenced by their size, it is important to
develop strategies in order to reduce the overall size of QD/
protein complexes. One effective route has been the use of
compact surface coatings which enable the direct binding of
+
2+
histag proteins to exposed or accessible Zn ions at the surface
2
3-26
of CdSe/ZnS QDs.
For this purpose, PEGylated dihydro-
Through application of QD-ND, we demonstrate the ability
to label and track histag proteins both in Vitro and in living
cells. In Vitro, we show evidence from dynamic light scattering
(DLS) and fluorescence correlation spectroscopy (FCS) mea-
surements that the QD-ND/protein complex is stable, small and
can be prepared with 1:1 stoichiometry following purification
by gel electrophoresis. To show specific binding in live cells,
we targeted a membrane receptor with an extracellular poly-
histidine sequence expressed in HeLa cells. Finally, we applied
our labeling strategy to record the movement of end binding
protein-1 (EB1), a microtubule associated proteins in Xenopus
cell extracts. EB1 is a 35 kDa cytoplasmic protein known to
specifically bind to microtubules and to regulate their dynam-
lipoic acid (DHLA) has been shown to facilitate the direct
1
8,27,28
interaction of histag proteins with the QD surface.
Using
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