From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 185

presence of the acceptor and the fraction of the interacting donor can be
easily deduced. We emphasize the fact that these calculations can be
performed during the time-lapse acquisition of the biosensor activity,
which makes it possible to follow over time the evolution of both the
FRET efficiency and the proportion of the interacting donor a 1 .

6. DESIGN AND OPTIMIZATION OF GENETICALLY
ENCODED KARs

The design of a FRET biosensor for imaging biochemistry in living
cells is based on the development of a single polypeptide capable of gener-
ating a conformational change that modulates FRET efficiency in response
to a biochemical event, such as phosphorylation in this context.
A genetically encoded KAR is composed of the following key elements:
an MRE consisting of a substrate peptide for the kinase of interest, a pho-
sphoamino acid-binding domain to detect the target activity, and a reporting
element consisting of a fluorescent protein-based FRET couple flanking the
sensing element. These functional parts are joined together with linkers
whose optimization is needed as they readily affect the overall dynamic range
of the biosensor ( Fig. 5.20 ).

...

...

...

CFP 14.3.3

...

mTurquoise

WD40 YPet

eCFP

FHA1 Venus Cytosol

CyPET WW CpVenus Nucleus

Cerule
an

Golgi

FHA2 YFP

Donor fluorescent
protein

Acceptor fluorescent
protein

Targeting sequence
(optional)

PAABD Substrate

...

(SAGG)n

EAAAR

GGSGGS

72 Gly

Linker

Figure 5.20 Necessary ingredients for making KARs.