From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 148

Biosensor

Molecular
Recognition
Element

Analyte

Transducer

Measurable signal

Figure 5.1 Schematic representation of the basic principle of the biosensor.

The first of a long line of such biosensors was developed in R. Tsien's lab
and reported in 1996. 1 While of simple design, it was the first to demonstrate
that fluorescent protein variants could be used for biosensing. This first ge-
netically encoded biosensor was composed of a blue fluorescent protein
(BFP) and a green fluorescent protein (GFP) encompassing a trypsin cleav-
able linker. Thereafter, many different types of biosensors have been devel-
oped with various molecular structures ( Fig. 5.2 ).
All genetically encoded fluorescent protein (FP)-based biosensors are clas-
sified into groups depending on their structure. 2,3 Kinase activity reporters
(KARs) are included in the group based on intramolecular F¨rster
resonance energy transfer (FRET). The archetypal structure of such
biosensors consists of two FPs flanking an MRE ( Fig. 5.3 ). The interaction
of an active kinase with its specific MRE (substrate

PAABD) leads to a
change in the molecular conformation of the MRE. This change alters the
distance and/or relative orientation between the two FPs and,
consequently, the FRET signal. This chapter focuses on genetically
encoded biosensors, and specifically those dedicated to kinase activity
measurements. Although kinase activity reporters (KARs) vary in specificity
depending on the choice of the substrate, the design strategy of these
reporters remains universal. 4
Genetically encoded FRET biosensors can be used to analyze molecular
events in single living cells and tissue, and even expressed in animals (e.g.,
aquatic animals, transgenic mice). They are expressed in the native context
of a living cell to report on dynamic events. However, as described by
Frommer et al. , 2 these FRET biosensors actively sense (“active reporters”)
cellular microenvironments and even the subcellular microenvironment
and cannot be regarded as “passive reporters” such as for instance, regular
FP-fused protein.

þ