Angewandte
Chemie
of 0.32 and 0.40, respectively.[21] Moreover, the complexes
exhibited similar brightness and lifetimes when conjugated to
peptides or proteins.[18,20] Therefore, we prepared heterodi-
mers of cs124-DTPA and cs124-TTHA linked to TMP using a
15-atom linker (Scheme 1), reasoning that the conjugation
would preserve the essential characteristics of the parent
complexes. Raymond and co-workers reported an extremely
bright (e ꢀ 28000mÀ1 cmÀ1 at 354 nm, quantum yield = 0.59)
multidentate 2-hydroxyisophthalamide (IAM) terbium che-
late.[22] We covalently linked TMP to a proprietary analogue
of the IAM complex (Lumi4) that has similar brightness and a
luminescence lifetime (ca. 2.7 ms) and remains unchanged
upon conjugation to proteins.[23] Each of the TMP-TCs that
we prepared exhibited characteristic terbium luminescence
when complexed with the metal (Supporting Information,
Figure S1).
For both in vitro and live-cell applications, TMP-TCs must
necessarily bind with high affinity to eDHFR fusion proteins.
To determine whether the TMP-TCs could bind to eDHFR
and serve as FRET donors to green fluorescent protein
(GFP), we titrated a purified eDHFR-GFP fusion protein
against a fixed concentration (20 nm) of the different TMP-
TCs. Using a time-resolved fluorescence plate reader, we
detected sensitized, long-lifetime (> 100 ms) emission of GFP
that increased with increasing protein concentration
(Figure 1). Addition of excess TMP substantially reduced
the signal, indicating that intramolecular TR-FRET occurred
between the eDHFR-bound conjugates and GFP. The relative
intensities of sensitized GFP emission at binding saturation
were positively correlated to the reported quantum yields of
the complexes. A nonlinear, least-squares fit of the data
showed the dissociation constants for binding to eDHFR of
TMP-cDTPA, TMP-cTTHA, and TMP-Lumi4 to be (9 Æ
1.3) nm, (22 Æ 3.0) nm, and (1.8 Æ 0.3) nm, respectively. The
measured affinities were higher than a previously reported
value for binding of a TMP-fluorescein conjugate to eDHFR
(KD ꢀ 30 nm)[17] and approach the value of free TMP.
We next sought to determine whether TMP-TCs could be
used to label eDHFR fusion proteins in living mammalian
cells. NIH3T3 fibroblast cells were transiently co-transfected
with two plasmid DNA vectors; one that expressed plasma
membrane targeted eDHFR and another that expressed
nucleus-localized cyan fluorescent protein (CFP), included as
a positive control for transfection. The cells were incubated in
growth medium containing 100 mm TMP-cTTHA for 20 hours,
washed, and imaged using an epi-fluorescence microscope
capable of pulsed UV excitation and time-resolved detection.
No specific labeling of plasma membrane-localized eDHFR
was observed in cells that expressed nucleus-localized CFP
(Figure 2a,b). Nonspecific luminescence was detected in all
cells, possibly indicating endocytosis of the compound and
trapping in lysosomes. When similar experiments were
performed with lower concentrations and or shorter incuba-
tion times, long-lifetime luminescence could not be detected
in cells incubated with any of the TMP-TCs.
While intracellular labeling of eDHFR with the TMP-TCs
was not possible, we were able to successfully label eDHFR
expressed on the cell surface. NIH3T3 fibroblasts were co-
transfected with the nucleus-localized CFP expression plas-
mid and a vector that expressed eDHFR on the extracellular
surface of the plasma membrane (pDisplay-eDHFR). 24
hours after transfection, the cells were incubated in growth
medium containing 1 mm TMP-Lumi4 for 10 minutes. Sub-
sequently, the cells were washed and imaged. A distinct
membrane luminescence was observed only in cells that
expressed nucleus-localized CFP when the cells were imaged
in time-resolved mode (Figure 2c,d). Owing to the dissocia-
tion of TMP-Lumi4 from eDHFR and diffusion into the
medium, the membrane fluorescence could only be detected
for approximately 20 minutes. A control experiment estab-
lished that the membrane fluorescence was dependent on the
specific labeling of the eDHFR fusion protein with TMP-
Lumi4. Pre-incubation of the cells expressing membrane-
targeted eDHFR in medium containing 10 mm TMP, followed
by incubation in medium containing 1 mm TMP-Lumi4
resulted in no membrane staining. We were only able to
detect cell-surface labeling of eDHFR with TMP-Lumi4, and
not with TMP-cDTPA or TMP-cTTHA.
Herein we have shown that the high-affinity noncovalent
interaction between TMP and eDHFR provides an effective
means for imparting terbium luminescence to recombinant
fusion proteins. TMP-TCs exhibited characteristic lumines-
cence and high affinity for eDHFR, and they proved to be
efficient sensitizers of GFP emission in an intramolecular TR-
FRET assay. TMP-Lumi4 was particularly effective, binding
to eDHFR-GFP with about 2 nm affinity and exhibiting more
than 100-fold increase in FRET signal upon binding satura-
tion. As FRET donors to GFP, TMP-TCs could be used to
detect interactions between eDHFR and GFP fusion proteins.
This would be particularly useful when protein-specific
antibodies are unavailable, or in situations where direct
conjugation of proteins with terbium complexes is problem-
atic, such as assays of cell lysates. As prepared, the TMP-TCs
reported herein are cell-impermeable, and can only be used to
Figure 1. Intramololecular, time-resolved, fluorescence resonance
energy transfer (TR-FRET) between eDHFR-bound TMP-TCs and GFP.
Increasing concentrations (C) of purified eDHFR-GFP were titrated
against a constant concentration (20 nm) of each compound. Sensi-
tized GFP emission (520 nm) was detected after a time delay of
100 ms, upon pulsed excitation with near-UV light (ca. 340 nm): TMP-
^
*
Lumi4 ( ), TMP-cTTHA ( ), TMP-cDTPA (ꢀ). Addition of 1 mm TMP
reduced the signal (TMP-cTTHA, &), confirming FRET. Lines represent
nonlinear least squares fit to the data.
Angew. Chem. Int. Ed. 2009, 48, 4990 –4992
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Rajapakse, Harsha E.
