phosphate-binding protein (PBP A197C) that is covalently modified with a diethylamino coumarin fluorophore via reaction of the
cysteine thiol with a maleimide group on the fluorescent molecule.
PBP has a two-lobed structure with the phosphate-binding site
located in between the two lobes (see Fig. 1a). P i binding induces
a large rigid body movement between the two sub-domains leading
to closure of the binding cleft. This brings the reporter fluorophore
into a different environment which, by change of specific interactions with the protein [4], causes a large increase in fluorescence
quantum yield (approximately eightfold) [5] and thus in measured
fluorescence intensity (see Fig. 1b). MDCC-PBP binds phosphate
rapidly (k on ¼ 1.36 Â 10
8 M
À1 s
À1 ) and tightly (K d ~ 0.1 μM,
values at pH 7.0 and 22
C) [1], properties which make it a very
sensitive probe for fast changes in P i concentrations.
There are several more recent variants of the original P i biosensor. The first, Rho-PBP, has two cysteines (one on each lobe, A17C
and A197C) modified with tetramethylrhodamine iodoacetamide
[6]. The P i -induced fluorescence change is based on a stacking
interaction of the two rhodamines [7, 8] which is altered upon P i
binding. Rho-PBP has very similar P i binding strength and dynamics to MDCC-PBP and is used in the same assay types. The main
differences between Rho-PBP and MDCC-PBP are the optical
properties of the fluorescent probe. Tetramethylrhodamine is
more photostable than diethylamino coumarin and has higher
excitation and emission wavelengths, properties that are advantageous when using high intensity light sources (e.g., lasers) or where
optical interference with other assay components is an issue (e.g.,
small molecule screening). Another variant, Rho-PBPw (weak
binding), is based on Rho-PBP with an additional mutation
(I27G) that weakens the binding of P i [9]. This version, as well as
a genetically encoded variant of PBP [10], has potentially different
applications, which are not described here.
Based on their design principle, the above P i biosensors are
classified as reagentless biosensors, which are built from a protein
scaffold that specifically binds the molecule of interest and a
reporter group (fluorophore) that generates an optical readout.
Reagentless biosensors have been developed for the detection of a
number of small molecule metabolites, e.g., ADP and GDP [11–
13], ATP [14], the ATP/ADP ratio [15, 16], glucose [17, 18], or
glutamate [17, 19], and many of them are based on periplasmic binding protein scaffolds [17–20]. Other detection systems
exist, such as coupled-enzyme assays (e.g., [21, 22]) or dye-based
assays, for example P i -detecting phosphomolybdate assays (e.g.,
malachite green) [23]. However, reagentless biosensors offer the
advantages of being simple, consisting of only one component, and
having a fast response time which is limited only by the rate of
binding and conformation change of the scaffold, generally being
in the order of sub-seconds rather than up to minutes, e.g., for
290
Simone Kunzelmann
cysteine thiol with a maleimide group on the fluorescent molecule.
PBP has a two-lobed structure with the phosphate-binding site
located in between the two lobes (see Fig. 1a). P i binding induces
a large rigid body movement between the two sub-domains leading
to closure of the binding cleft. This brings the reporter fluorophore
into a different environment which, by change of specific interactions with the protein [4], causes a large increase in fluorescence
quantum yield (approximately eightfold) [5] and thus in measured
fluorescence intensity (see Fig. 1b). MDCC-PBP binds phosphate
rapidly (k on ¼ 1.36 Â 10
8 M
À1 s
À1 ) and tightly (K d ~ 0.1 μM,
values at pH 7.0 and 22
C) [1], properties which make it a very
sensitive probe for fast changes in P i concentrations.
There are several more recent variants of the original P i biosensor. The first, Rho-PBP, has two cysteines (one on each lobe, A17C
and A197C) modified with tetramethylrhodamine iodoacetamide
[6]. The P i -induced fluorescence change is based on a stacking
interaction of the two rhodamines [7, 8] which is altered upon P i
binding. Rho-PBP has very similar P i binding strength and dynamics to MDCC-PBP and is used in the same assay types. The main
differences between Rho-PBP and MDCC-PBP are the optical
properties of the fluorescent probe. Tetramethylrhodamine is
more photostable than diethylamino coumarin and has higher
excitation and emission wavelengths, properties that are advantageous when using high intensity light sources (e.g., lasers) or where
optical interference with other assay components is an issue (e.g.,
small molecule screening). Another variant, Rho-PBPw (weak
binding), is based on Rho-PBP with an additional mutation
(I27G) that weakens the binding of P i [9]. This version, as well as
a genetically encoded variant of PBP [10], has potentially different
applications, which are not described here.
Based on their design principle, the above P i biosensors are
classified as reagentless biosensors, which are built from a protein
scaffold that specifically binds the molecule of interest and a
reporter group (fluorophore) that generates an optical readout.
Reagentless biosensors have been developed for the detection of a
number of small molecule metabolites, e.g., ADP and GDP [11–
13], ATP [14], the ATP/ADP ratio [15, 16], glucose [17, 18], or
glutamate [17, 19], and many of them are based on periplasmic binding protein scaffolds [17–20]. Other detection systems
exist, such as coupled-enzyme assays (e.g., [21, 22]) or dye-based
assays, for example P i -detecting phosphomolybdate assays (e.g.,
malachite green) [23]. However, reagentless biosensors offer the
advantages of being simple, consisting of only one component, and
having a fast response time which is limited only by the rate of
binding and conformation change of the scaffold, generally being
in the order of sub-seconds rather than up to minutes, e.g., for
290
Simone Kunzelmann