Structure–Function Relationships in Fluorescent Marker Proteins of the Green Fluorescent Protein Family - Fluorescent Proteins: From Understanding to Design - page 235

2.3 Variations of the Chromophore Motif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
2.4 Quaternary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
3 Protein Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
3.1 Maturation and Thermotolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
3.2 Cellular Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
3.3 Monomerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
3.4 Photostability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
3.5 Color Tuning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

1 Introduction

The green fluorescent protein (GFP) and the related fluorescent proteins (FPs) of the
GFP family have revolutionized life sciences research by enabling a vast array of
novel approaches to study biomolecular processes, especially in living cells and
organisms. In recognition of GFP's enormous impact on the life sciences, the Nobel
Committee for Chemistry awarded the Prize in 2008 to Osamu Shimomura, Martin
Chalfie, and Roger Y. Tsien “for the discovery and development of the green
fluorescent protein, GFP” [ 1 ]. Shimomura isolated the protein already in 1962
from tissue extracts of the jellyfish Aequorea victoria [ 2 ]. But only in 1994, when
the GFP gene was expressed recombinantly in Escherichia coli and Caenorhabditis
elegans , researchers realized that GFP can be used as a genetically encoded fluores-
cence marker because the fluorophore forms spontaneously in a reaction that only
requires O 2 [ 3 , 4 ]. To extend its range of applications, color variants of A. victoria
GFP were developed by mutagenesis, with emission peaks ranging from blue (BFP)
to yellow (YFP) [ 5 ]. A major step forward was the discovery of GFP homologues in
anthozoa animals [ 6 - 8 ], so that a large number of fluorescent proteins (FPs) became
available as potential fluorescent marker tools including the long-sought orange and
red FPs. They extended the color palette for multicolor labeling or FRET experi-
ments, and were also highly welcomed for live-cell and tissue imaging applications
because of the reduced cellular autofluorescence and scattering in the red spectral
range. In recent years, so-called photoactivatable or optical highlighter FPs have
emerged as powerful new tools for cellular imaging [ 9 - 12 ]. Upon irradiation with
light of specific wavelengths, these FPs can either be switched reversibly between
a fluorescent and a nonfluorescent state (photoswitching), or they change their
fluorescence emission intensity or color irreversibly (photoconversion).
However, FPs obtained from natural sources frequently show poor performance
as fluorescence markers, including a tendency to aggregate or oligomerize, incom-
plete (or even completely lacking) chromophore formation (maturation), especially at
physiological temperatures (37 C), fluctuating emission (photodynamics, flickering),
and fast photobleaching. Detailed structural and spectroscopic investigations of GFP-
like proteins have furthered our understanding of structure-dynamics-function rela-
tionships in recent years up to a point that, in many instances, rational development
of optimized variants using genetic engineering approaches has become feasible.