Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 241

Peptides and polypeptides offer several major advantages inherent to
their nature. They can be readily produced through synthetic chemistry
or recombinant protein engineering; are easy to handle, store, and charac-
terize; and can further be modified with a wide variety of unnatural substit-
uents, such as modified amino acids, to improve specificity and selectivity, as
well as small synthetic fluorescent probes. Peptides are very small yet can
serve as substrates, docking sequences, or complementary biomolecular rec-
ognition interfaces, thereby offering a wide array of possibilities and strong
potential for development of fluorescent biosensors. Peptides also constitute
scaffolds for a wide variety of technological improvements, including intro-
duction of quenchers or caging of specific amino acids. Moreover, compared
to antibodies, they are cheaper to produce, display low antigenicity, and are
rapidly eliminated by the organism, thereby generating relatively low cyto-
toxicity. Peptide biosensors are readily applicable in vitro and in cell lysates.
Their applicability in living cells and in vivo , however, remains challenging,
requiring suitable methods to facilitate their intracellular delivery. Notwith-
standing, major advances in the field of protein and peptide delivery over the
past 20 years have provided efficient means of introducing this class of bio-
molecules into living cells and in vivo based on protein transduction domains
and cell-penetrating peptides (CPPs). 107,108 Once the issue of delivery is
solved, peptide biosensors offer a major advantage over genetically
encoded biosensors, in that they allow for immediate and controlled use,
compared to genetically encoded biosensors, which require long periods
of time for their expression and/or maturation ( Table 6.1 ).
Several strategies have been employed to design and engineer peptide
biosensors of kinase activity, which are quite different from the strategies de-
veloped to generate genetically encoded KARs. In all cases, phosphorylation
induces changes in the spectral properties of the fluorophore(s) incorporated
into the peptide scaffold, which may involve a shift in its emission wave-
length and/or an important change in its quantum yield. Fluorescent peptide
biosensors can be broadly divided into three different groups, depending on
the mechanism through which fluorescence reports on phosphorylation: en-
vironmentally sensitive biosensors, chelation-enhanced fluorescence
through metal-ion binding, and biosensors involving quenching/
unquenching strategies. We will describe the different categories of fluores-
cent peptide biosensors that have been developed to probe kinase activities,
together with some representative examples to illustrate their mechanism of
action and their applications (summarized in Table 6.3 ). Additional details