Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 317

a green GFP-like chromophore intermediate 21 has been recently revised.
According to the current understanding, the maturation pathway of the
DsRed-like red chromophore includes accumulation of a blue-emitting in-
termediate 22,23 possessing a structure thought to be similar to that of the
mTagBFP chromophore, a blue derivative of DsRed. These properties
have been utilized to generate fluorescent timers (see Section 3.1 ).

2.3. Circular permutation
The intricate posttranslational self-modification required for chromophore
formation suggests that major rearrangements or insertions within GFP would
prevent fluorescence emission. However, the three-dimensional structure of
GFP ( Fig. 8.1A ) implies that there is still plenty of room for manipulation in
the loop regions between the -sheets. Indeed, Dr. Tsien and colleagues have

succeeded in engineering several fluorescent rearrangements of GFP and other
fluorescent proteins in which the amino and carboxyl portions are inter-
changed and rejoined, with a short spacer (GlyGlyThrGlyGlySer) connecting
the original termini. 24 It is noteworthy that all interrupted positions are
located in the original C-terminal half of enhanced GFP ( Fig. 8.1A ). In
addition, some sites (His148, His169, and Ara227) are in

b

-strand segments,
whereas others are in the loops, as expected ( Fig. 8.1A ). More importantly,
certain residues within GFP, including Tyr145, tolerate insertion of other
proteins, and conformational changes in the insert profoundly influence the
fluorescence intensity. 24 These properties are utilized in the development
of single-fluorescent protein-based biosensors (see Section 3 ). Circular per-
mutations also alter the relative orientation of the chromophore to a fusion
partner, and this property is exploited in the improvement of biosensors based
on the principles of FRET (F¨rster resonance energy transfer; see Section 5 ).

b

2.4. Fluorescent proteins as tags
The discovery and cloning of GFP and its relatives have undoubtedly
advanced our understanding of basic biology. For example, use of fluores-
cent proteins has facilitated routine monitoring of gene activation as well as
the selective labeling and analysis of single proteins, cellular organelles, and
even whole cells. 6,13
Tracking cellular proteins in vivo with fluorescent tags was made routine
by the development of GFP and its family members. Fluorescent proteins are
usually appended to the amino or carboxyl terminus of the host protein.
The choice of carboxyl versus amino terminal attachment can be guided