Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 322

changes its excitation peak from 440 nm (protonated neutral chromophore
of mKeima) to 585 nm (anionic chromophore) in response to
acidification, thereby enabling pH monitoring by dual-excitation, single-
emission observation. 45 Introduction of a His148Gln mutation into
the yellow-emitting mutant of GFP (YFP) makes YFP a halide ion
sensor. 51 The fluorescence intensity of this mutant YFP is sensitive to the
concentrations of F - ,Cl - ,Br - ,I - ,NO 3 ,SCN - ,ClO 4 , and formate, in
addition to pH. 51 Voltage-sensitive fluorescent proteins (VSFPs) have also
been developed to allow optical measurement of membrane potential. 52-55
By tagging Citrine, a variant of CFP, to the carboxyl terminus of a Ciona
intestinalis voltage-sensor-containing phosphatase (Ci-VSP) via a short
linker, a biosensor that reports fluorescence activation kinetics closely
resembling the fast “gating” currents (VSFP3.1) has been developed,
although the detailed mechanism through which the emission intensity is
modulated is still lacking.
Ca 2 þ sensors, which include camgaroo1, 24 pericam, 56 G-CaMP, 57
Case12, 38 and GECO, have also been well studied. 58 These biosensors,
which result from the insertion of Ca 2 þ -sensing proteins into circular
permutated fluorescent proteins, enable researchers to visualize elevation
of intracellular Ca 2 þ by increases in intensity of a single spectrum. Such bio-
sensors have great advantages over FRET-based Ca 2 þ biosensors such as
cameleon 59 and FIP-CB SM 60 because of their narrowed emission wave-
length ranges and enhanced signal-to-noise ratios.
With the appropriate insertions, circularly permutated fluorescent pro-
teins can detect changes in a range of parameters. Dr. Lukynanov and col-
leagues developed a fluorescent biosensor that is highly specific for hydrogen
peroxide (H 2 O 2 ) via insertion of circularly permuted YFP into the regula-
tory domain of the prokaryotic H 2 O 2 -sensing protein OxyR (HyPer). 61
A biosensor for adenylate nucleotides has also been generated from a circu-
larly permuted variant of GFP and the ATP-binding bacterial protein GlnK1
(from Methanococcus jannaschii ). 62
4. BIMOLECULAR FLUORESCENCE COMPLEMENTATION
A range of proteins can be divided into two (or more) fragments that
individually possess no function but that can be reconstituted as a function-
ally active complex when they are brought in proximity to each other.
Fusing the fragments to partners that interact with each other enhances
the efficacy of their assembly. Therefore, the activity of the complex can