One-Pot Construction of Fluorescent Biosensors
A R T I C L E S
site (i.e., in most cases, restricted to tagging at the N- and
C-terminus of proteins) severely limit the range of scaffold
candidates that the FP-fusion strategy can be applied to. The
construction of FP-based biosensors has so far been successful
only when the fusion constructs induce a large conformational
change upon binding to the analyte. Such cases are unfortunately
rather rare.
of a cysteine residue, followed by the use of thiol chemistry to
attach an organic dye.2,7 However, many proteins are not
amenable to this strategy because of the presence of an inherent
cysteine residue(s). The acquisition of mutant proteins which
retain natural ligand-binding properties (affinity and specificity)
is not always successful. Although we have developed the
post(photo)affinity labeling modification methods as tools for
introducing fluorescent molecules into natural proteins without
genetic manipulation, these approaches require multiple chemi-
cal reactions to be performed on the protein surfaces.8 In
addition, all the prevalent protein modification procedures9
require a careful purification step after the labeling reaction and
cannot be applied to crude protein mixtures. Clearly, the
establishment of a new methodology that can overcome the
aforementioned limitations is of considerable importance for
the progression of semisynthetic biosensors.
Here we describe a novel, rational protein engineering strategy
that allows one-step construction of turn-on fluorescent semi-
synthetic biosensors. The method is based on the recently
developed ligand-directed tosyl (LDT) chemistry, which is
capable of labeling specific proteins with diverse synthetic
probes with high site-specificity and target-selectivity.10 By
designing a new class of labeling reagents, quenched ligand-
directed tosylate (Q-LDT) compounds, it was feasible to convert
natural proteins to fluorescently labeled proteins that can be
directly used as biosensors based on the bimolecular fluores-
cence quenching and recovery (BFQR) mechanism (Figure 1).11
We successfully applied the strategy to two proteins, carbonic
anhydrase II (CAII) and the Src homology 2 (SH2) domain, to
generate turn-on fluorescent biosensors toward the inhibitors
and phosphorylated peptides, respectively, by a single labeling
step not only in a purified form but also in a bacterial cell lysate.
Site-specific chemical modification of proteins with synthetic
fluorescent dyes is another important means to afford semisyn-
thetic, fluorescent biosensors.2,7,8 The major strength of this
chemistry-driven approach is the flexibility in terms of the choice
of fluorophore and the attachment site (not restricted at the
termini of proteins). Consequently, this approach essentially
permits various fluorescence transduction mechanisms to be
integrated into protein frameworks in a tailor-made manner.
During the past decades, a number of semisynthetic biosensors
have been constructed, particularly by installing an environment-
sensitive dye into a protein scaffold at a specific site such as
the periphery of the analyte-binding pocket.7,8 These biosensors
have been shown to transduce the analyte-binding event into a
change in the fluorescence intensity and/or wavelength by
sensing a subtle change of the microenvironment surrounding
the fluorophore, such as pH and solvent polarity. Importantly,
these examples have clearly demonstrated that the semisynthetic
strategy is powerful and has the potential to be applicable in
the conversion of nearly all possible protein scaffolds to
corresponding biosensors. However, there are challenges to be
addressed. First, it remains difficult to rationally predict the
appropriate site and type of fluorophore to be introduced to
achieve the optimal fluorescence response. In this regard,
fluorescence enhancement, rather than reduction, is favorable
for more sensitive and accurate detection. Second, even after
the optimization through a laborious trial-and-error process, in
many cases, the signal change is small to moderate. Third, as a
more fundamental problem, methods for site-specific protein
modification are limited. Most of the semisynthetic biosensors
previously reported were prepared though genetic incorporation
Results and Discussion
General Design of Q-LDT Compounds. Our idea to construct
turn-on fluorescent biosensors is illustrated in Figure 1. The
concept is based on the LDT chemistry that we recently
developed and is a new type of affinity-guided protein surface
labeling scheme.10 In contrast to existing affinity labeling
reactions,12,13 the LDT-based approach is unique in that the
ligand moiety is cleaved concomitantly with the labeling process,
thus enabling covalent, selective attachment of probes of interest
to specific target proteins without irreversibly abolishing the
function of the labeled proteins. By taking advantage of this
strategy, we designed a new class of protein labeling reagents,
Q-LDT compounds, which contain a fluorescent dye and its
quencher together so that the labeling reagent itself is only
weakly fluorescent. The quencher is introduced as a part of the
leaving group. Upon binding of the ligand moiety to the target
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