Biology Reference
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peptide biosensors. The discovery of the green fluorescent protein (GFP) led
to the development of genetically encoded reporters, which have been suc-
cessfully applied to the study of protein kinase behavior in living cells with
high spatial and temporal resolution (for review, see Refs.
32-36
). In
parallel, combined efforts in fluorescence chemistry and in chemical
biology have led to the design of an entirely different family of biosensors,
based on peptide, polypeptide, or polymeric scaffolds, onto which small
synthetic probes with particularly attractive photophysical properties can be
incorporated. These nongenetic biosensors have been applied to monitor
protein kinase activities
in vitro
and in more complex biological samples,
including cell extracts and living cells, with an equally successful outcome
(for review, see Refs.
35-38
). While biosensors from each of these classes
exhibit specific advantages, they equally suffer from characteristic drawbacks
and shortcomings, which will be discussed further below and are
summarized in
Table 6.1
. Genetically encoded biosensors are user-friendly,
allowing for easy manipulation and transfection into cells. However, most of
these biosensors are based on changes in fluorescence resonance energy
transfer (FRET) between two AFPs, which are often fairly weak compared
to the fluorescence changes associated with nongenetic, environmentally
sensitive biosensors. In contrast, whereas peptide and protein biosensors are
easy to engineer and handle
in vitro
, it is difficult to introduce these into
living cells and
in vivo
. However, they offer complete control over the
concentrations used and the timing of their availability
in cellulo
,whereas
genetically encoded biosensors are heterogeneously expressed both in terms
of levels and timing.
2. GENETICALLY ENCODED REPORTERS OF
PROTEIN KINASES
The discovery of the GFP and its engineering into a wide variety of
AFPs prompted the development of GFP-based reporters and the design
of genetically encoded biosensors, which were rapidly applied to the field
of protein kinases.
32-36,39-45
Several groups of genetically encoded
biosensors have been developed (for review, see Refs.
34,44
): (i) single-
chain biosensors, which bear a pair of FRET AFPs within the same
molecule that are brought together and undergo energy transfer due to
intramolecular conformational changes in response to the recognition
event; (ii) two-chain biosensors, in which two AFPs lie on two different
molecules capable of interacting and undergoing intermolecular FRET
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