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properties than ensemble measurements can hope to provide. A particular strength
of single molecule studies is the ability to identify rare forms and subensembles
that are hidden or easily overlooked in ensemble studies, and to visualize the
evolution of these forms in time, which is impossible in ensemble studies. To access
the details of the photophysical complexity of VFPs, single molecule studies are of
great value. In particular, many aspects of the spectral versatility of fluorescent
proteins can only be analyzed on the single molecule level using spectrally resolved
single molecule spectroscopy.
2 Basis for Photophysical Complexity of VFPs
VFPs show an extraordinary spectral versatility and photophysical complexity
compared to most commonly used chemical marker molecules. The reasons for
this spectral complexity are manifold and have their origin, on the one hand, in the
general structure of VFPs and, on the other hand, in the details of the formation of
the light-emitting chromophore.
All VFPs now known have been isolated from different marine life forms or
were created by using the toolbox of protein engineering to modify these proteins
found in nature. Remarkably, all these proteins share the same basic structure
elucidated for Aequoria GFP [ 37 , 38 ]. GFP has a molecular weight of 26.9 kDa
and forms a barrel-like structure with a diameter of about 2.4 nm and a height of
4.2 nm (Fig. 1 ). 11
-sheets form the outer wall of the barrel structure, while an
-helix runs diagonally through this barrel. The fluorescent chromophore is
enclosed in the center of the
The formation of this light-emitting chromophore is a complex, multistage chemi-
cal reaction within the protein (see Fig. 1 ), with significant potential for the formation
of side products that complicate the emission behavior of the VFPs, e.g., by resulting
in a mixture of different end product chromophores of different emission colors [ 19 ,
40 , 43 , 44 ]. A prime example of the parallel formation of different chromophores
starting from identical precursors is the tetrameric reef coral fluorescent protein
DsRed, in which a green- or a red-emitting chromophore can be formed [ 40 ].
Variants generated from this protein show a very different spectral appearance,
depending on the exact ratio of occurrence of these two chromophores within the
tetramer. From a chemistry point of view, the appearance of different end products in
a complex chemical reaction over many steps is not unusual, and is in fact expected.
When synthesizing chemical marker chromophores, this problem of unwanted side
products is solved by a number of purification steps that result in essentially pure
emitter molecules of choice. Clearly, this approach cannot be followed using geneti-
cally encoded fluorescent markers such as the VFPs that are created in vivo. As a
result, when using VFPs as marker molecules, one has to accept the presence of
different end product chromophores that are formed within the proteins.
In addition to the spontaneous formation of different chromophores, a number of
photoinduced modifications of the chromophore and its nanoenvironment have
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