Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 330

(in the absence of protein cleavage or degradation), making the emission
ratio a reliable FRET index. In other words,

YFP ¼ c CFP : ½ 8 : 15

When combined with Eq. (8.14) , this becomes

FRET
CFP ¼

½ FRET c þ a CFP þ b ð c CFP Þ

CFP

FRET c
CFP þ

¼ ð a þ bc Þ: ½ 8 : 16

On the other hand, obtaining “good” intramolecular FRET-based
biosensors that are well suited for practical use is a major barrier to the
use of this method. A number of biosensors that enable visualization of cel-
lular events under physiological conditions have been developed and have
been summarized in several excellent reviews 83,95 ; thus, we will refrain
from discussing each individual biosensor. Much work has been done in
an effort to construct better biosensors than current biosensor versions.
A rational trial to improve the properties of biosensors involves
introduction of circular permutations into one or both of the donor/
acceptor fluorescent proteins. D .Miyawakiandcoleague ,who
originally developed the FRET-based Ca 2 þ biosensor Cameleon and its
derivate yellow Cameleon (YC), have succeeded in generating an
improved YC by conducting circular permutation on Venus, the
acceptor of YC. 96 The improved YC3.60, which possesses cp173 Venus
(starting its new amino terminus at Asp173), is equally bright but shows
a five- to sixfold larger dynamic range compared with YC3.12 with
“wild-type” Venus. We have also improved the CrkL-based biosensor 5
using a similar strategy in which circular permutation was conducted on
the donor fluorescent protein, ECFP (see Section 7 ). In addition, a
similar approach using multiple cpCFPs and cpYFPs was used to
enhance the dynamic range of a troponin C (TnC)-based Ca 2 þ
indicator, TN-L15. 97,98 Despite these achievements, it remains unclear
whether it is the distance between or the relative orientation of the
chromophores that is responsible for the improved dynamic ranges.
Thus, an enormous number of experiments to test such constructs are
still required to generate improved combinations of donors and
acceptors for each FRET biosensor.
Another rational strategy for the improvement of FRET constructs
is fine-tuning of the linker regions within the biosensors. For example,