A red Cy3-based biarsenical fluorescent probe targeted to a complementary binding peptide

Article abstract of DOI:10.1021/ja070003c

We have synthesized a red biarsenical fluorescent probe, AsCy3, with good photostability, low pH sensitivity, high absorbance, and good quantum yield. It is directed specifically to a small tetracysteine peptide binding motif, Cy3TAG (CysCysLysAlaGluAlaAlaCysCys), in the presence of other tetracysteine tags. This new probe provides a FRET partner to biarsenical dye FlAsH, making this discovery an important step toward a whole toolkit of colored probes directed to different small peptide motifs. Copyright

Full text of DOI:10.1021/ja070003c

Published on Web 06/22/2007
A Red Cy3-Based Biarsenical Fluorescent Probe Targeted to a
Complementary Binding Peptide
Haishi Cao, Yijia Xiong, Ting Wang, Baowei Chen, Thomas C. Squier, and M. Uljana Mayer*
Cell Biology and Biochemistry Group, Pacific Northwest National Laboratory, Richland, Washington 99352
Received January 1, 2007; E-mail: uljana.mayer@pnl.gov
Small-molecule biarsenical multiuse affinity probes (MAPs)
FlAsH and ReAsH,1,2 in conjunction with complementary protein
tags, are important new tools for analyzing cellular function through
live-cell imaging,3,4 targeted protein inactivation,5 and the measure-
ment of protein dynamics and binding.6 In addition, MAPs serve
as affinity reagents for isolating intact protein complexes for
complementary structural measurements.7 These first-generation
MAPs bind to a tetracoordinate arsenic group (TAG) binding motif
(i.e., CCXXCC or FlAsHTAG) genetically engineered onto a
protein of interest. They are superior to other targeted labeling
strategies (such as the Halo-tag, the SNAP tag, and fluorescent
proteins) in that the small peptide tag does not disrupt protein-
protein interactions nor perturb the correct trafficking of tagged
proteins.8,9 The conserved interatomic distance (∼6 Å) between the
two arsenic moieties in FlAsH and ReAsH complicates the selective
labeling of multiple proteins with different reporters. To overcome
these limitations, we have synthesized a new biarsenical MAP (i.e.,
AsCy3) based on Cy3, a member of the cyanine dye family, whose
well-recognized brightness and photostability facilitate their utility
in single-molecule measurements. The large interatomic distance
between the two arsenics in AsCy3 (∼14.5 Å) coupled with the
identification of a complementary high-affinity binding sequence
CCKAEAACC (Cy3TAG) permits the simultaneous application of
both AsCy3 and FlAsH to selectively label their respective binding
TAGs in different proteins. In addition, the fluorescence of FlAsH
overlaps with the absorption of AsCy3, which can act as an acceptor
of fluorescence resonance energy transfer (FRET) to allow ratio-
metric measurements of protein association.
Figure 1. Left Panel: Identical absorbance spectra (- - -) and fluorescence
emission spectra of AsCy3-EDT2 (- ‚ -) or the AsCy3-labeled Cy3TAG on
the CaM-binding smMLCK peptide (s). Right Panel: Fluorescence
enhancement of AsCy3 (1 µM) associated with addition of Cy3TAG on
sm-MLCK (b) or control, deficient in Cy3TAG, sm-MLCK peptide (O).
Scheme 1. Synthesis of AsCy3
Synthesis of AsCy3 involved four steps (Scheme 1). Briefly,
sulfonated Cy310 was prepared according to the procedure of Li et
al.,11 followed by mercuration and transmetalation to arsenic, which
was liganded to ethanedithiol (EDT) for enhanced stability.2,14 The
overall yield of AsCy3 was 38%. Using 2D NMR to determine the
structure of AsCy3 shows that the mercuration of the cyanine
scaffold is specific with metal insertion para to nitrogen of the
indoline ring system. Alternative synthetic routes, such as attaching
the arsenic to the cyanine subunits prior to creating the linker region
or coupling of the indolines under basic condition, resulted in
substantially lower yields.
The inter-arsenic distance in AsCy3 (14.5 Å) was used in the
design of a Cy3TAG binding sequence. On the basis of in silico
distance measurements on NMR structures of R-helices12 and
hairpin-connected â-sheets, five different peptides were designed,
and high-affinity binding was demonstrated for the Cy3TAG
sequence CCKAEAACC (Figure 1, right panel). This sequence
places two cysteine pairs two helical turns (i.e., ∼14 Å) apart, while
the optimal FlAsH binding motif was based on one helical turn
and is improved by the closer spacing provided by the proline-
glycine turn.2,13 Binding of AsCy3 is rapid and occurs in <15 s
(see Supporting Information Figure S1). In comparison, FlAsH and
ReAsH binding under similar conditions occurs in a time scale of
several minutes.14
The direct equimolar association between AsCy3 and the
Cy3TAG can be fit assuming a simple Langmuir binding curve
(Figure 1 and Supporting Information Figure S2). AsCy3 binds to
the Cy3TAG with high affinity (apparent Kd ) 80 ( 10 nM) in
the presence of 100 µM EDT, which is commonly used to reduce
nonspecific binding, when labeling tagged proteins in cells.13 The
observed binding affinity of AsCy3 to the Cy3TAG is comparable
to that for the association between FlAsH and FlAsHTAG in the
presence of similar amounts of EDT,27 whose reported dissociation
constant in the absence of added EDT is ∼10 pM.2,13
AsCy3 has absorbance and fluorescence emission maxima at 560
and 568 nm, respectively. The absorbance spectrum is insensitive
to AsCy3 binding to the Cy3TAG and the release of EDT, while
the fluorescence spectrum is red-shifted (λmax ) 576 nm) with a
concomitant 6-fold increase in the fluorescence quantum yield (Q
) 0.28) (Figure 1, left panel). The peptide-bound AsCy3 conjugate
has enhanced photostability in comparison to the corresponding
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10.1021/ja070003c CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
purpose,16 we believe that the new AsCy3 platform will perform
as well as the first generation of biarsenicals for in vivo imaging
and extend their utility to two-color measurements of protein
complexes whose function is disrupted by large tags.
In conclusion, we have synthesized a new biarsenical fluorescent
probe, AsCy3, that in conjunction with a high-affinity binding motif
(i.e., Cy3TAG) (CCKAEAACC) permits specific labeling of the
Cy3TAG in the presence of the previously identified FlAsHTAG
(CCXXCC). Further, AsCy3 provides a FRET partner to the
biarsenical dye FlAsH, permitting measurements of protein-protein
interactions. AsCy3 has superior photostability and a minimal
environmental sensitivity compared to the existing biarsenical
probes FlAsH and ReAsH. Thus, the discovery of the new
biarsenical probe AsCy3 provides an important next step in
developing a whole toolkit of colored probes directed to different
small binding motifs.
Figure 2. Left Panel: Fluorescence emission spectra of FlAsH-labeled
CaM (1 µM) titrated with AsCy3-labeled smMLCK peptide from 0 to 1.0
µM in 0.2 µM increments. Right Panel: Labeling specificity of the
FlAsHTAG in CaM and Cy3TAG in smMLCK reacted first with AsCy3,
then FlAsH (all 1 µM). Same SDS-PAGE lane was imaged to detect AsCy3
(red) and FlAsH (green).
Acknowledgment. We thank Ping Yan for synthesizing the
FlAsH-EDT2. This research was supported by the Genomics: GTL
program (Grant 45701) of the U.S. Department of Energy. PNNL
is operated for the DOE by Battelle Memorial Institute under
Contract DE-AC05-76RL0 1830.17
peptide conjugates involving FlAsH and ReAsH, retaining optimal
spectral intensity for extended periods of time; in comparison to
FlAsH and ReAsH, bleaching requires 3- and >30-fold more light
intensity, respectively.14 The fluorescence of AsCy3 is pH-
independent between 4 and 9 (data not shown), permitting accurate
ratiometric measurements in applications involving FRET between
FlAsH and AsCy3 at neutral pH. Further, the large extinction
coefficient (ꢀ) of 180 000 M-1 cm-1 yields a brightness (ꢀ × Q )
5.0 × 104 M-1 cm-1) that is similar to that of FlAsH (4-6 × 104
Supporting Information Available: Experimental procedures for
the synthesis of AsCy3, FRET and labeling experiments, expanded
figures for binding and kinetics, and further references. This material
is available free of charge via the Internet at http://pubs.acs.org.
M-1 cm-1 2,13
and considerably higher than the red probe ReAsH
)
References
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