Biomedical Engineering Reference
In-Depth Information
Although this simple amino acid motif is commonly found throughout in
nature, it does not generally result in fluorescence. What is unique to the
fluorescent protein is that the location of this peptide triplet resides in the
center of a remarkably stable barrel structure consisting of 11
β
-sheets folded
into a tube (Fig. 10.3). Since 1992, the FPs have become widely used as non-
invasive markers in living cells, and their successful integration into variety
of living systems illustrates that the expression of these proteins in cells is
well tolerated. Virtually, any protein can be tagged with GFP, the resulting
chimera often retains parent-protein targeting and function when expressed
in cells, and therefore can be used as a fluorescent reporter to study protein
dynamics.
The Future of Fluorescent Proteins
The GFP has been engineered to produce a vast number of variously colored
mutants, fusion proteins, and biosensors that are broadly referred to as FPs.
The GFP sequence has been modified for optimizing the expression in different
cell types as well as the generation of GFP variants with more favorable
spectral properties, including increased brightness, relative resistance to the
effects of pH variation on fluorescence, and photostability. References [1, 3]
illustrate in detail the biochemical and fluorescent properties of GFP-variants.
The scientist who mostly contributed to understand how GFP works and
developed new techniques and mutants is Roger Tsien. His group has obtained
mutants that start fluorescing faster than wild type GFP, are brighter and
have different colors (Fig. 10.4) [3].
The latest generation of jellyfish variants has solved most of the deficien-
cies of the first generation FPs. The search for a monomeric, bright, and
fast-maturing red FP has resulted in several new and interesting classes of
FPs, particularly those derived from coral species. Development of existing
FPs, together with new technologies, such as insertion of unnatural amino
acids, will further expand the color palette. The current trend in fluorescent
probe technology is to expand the role of dyes that fluoresce into the far red
and near infrared. In mammalian cells, both autofluorescence and the absorp-
tion of light are greatly reduced at the red end of the spectrum. Thus, the
development of far red fluorescent probes would be extremely useful for the
examination of thick specimens and entire animals. Given the success of FPs
as reporters in transgenic systems (Fig. 10.5), the use of far red FPs in whole
organisms will become increasingly important in the coming years. Finally,
the tremendous potential in FP applications for the engineering of biosensors
is just now being realized. The success of these endeavors certainly suggests
that almost any biological parameter will be measurable using the appropriate
FP-based biosensor.
