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The properties of different subensembles and the transitions between these forms
were first addressed for ensembles of GFP group proteins using low temperature
spectroscopy. At cryogenic temperatures, three spectroscopically different, inter-
convertible forms were found for a number of GFP variants [ 48 , 80 , 81 ]. These
forms were named A-form with neutral chromophore, B-form with deprotonated,
anionic chromophore, and I-form which was found to be an intermediate involved
in the transition between these forms. The B-form with anionic chromophore is
strongly fluorescent and the predominant form in brightly emitting proteins such as
the Enhanced Yellow Fluorescent Protein (EYFP). Experiments at cryogenic tem-
peratures can establish limits on the heights of the energy barriers between the
various forms, but it is not possible to conclude from these experiments if the
different forms are of relevance at room temperature conditions typical for the use
of these proteins.
To address the question of spectrally different forms, spectrally resolved single
molecule detection was used to analyze the fluorescent protein EYFP in detail [ 36 ]
by recording a large number of single protein spectral series. Once again the
emission maximum peak position was the chosen analysis parameter, and was
determined for each spectrum and assembled into the histogram presented in Fig. 6 .
Fig. 6 Characteristic single protein emission spectra of all spectrally different forms observed for
the enhanced yellow fluorescent protein (EYFP); ensemble spectrum in solid grey. The distribu-
tion of emission maximum positions from all analyzed single EYFP molecules is clearly not
unimodal. Besides the main distribution around 527 nm, side maxima on the blue as well as on the
red side of the main distribution are clearly discernible (Adapted from [ 36 ], with permission from
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