Most GFP-like fluorescent proteins exhibit small Stokes shifts (10–45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.

It is well known in spectroscopy of complex organic molecules that the large width of their absorption and emission bands is largely due to the existence of a continuous set of vibrational sublevels in each electronic state. The spectroscopic properties of dye molecules in solution are in addition influenced by the surrounding medium.(1-3) For several decades it had been believed that because of the fast energy exchange between the vibrational sublevels the fluorescence spectrum of organic dye in solution was independent of the frequency of the exciting light. In 1970 it was shown (4) for the first time that apart from molecular vibrations there is another cause of the substantial broadening of electronic spectra of organic molecules in solution, namely, the fluctuations of the structure of the solvation shell surrounding the molecule. The variation of the local electric field caused by the fluctuation of the shell structure leads to a statistical distribution of the frequencies of the electronic transitions of the molecules and, therefore, to inhomogeneous broadening of the dye spectrum.This broadening was experimentally demonstrated (4)(5)(6) from the dependence of the fluorescence spectrum of a dye solution on the exciting radiation frequency at 77°K. Later, Personov et al.(7) observed inhomogeneous broadening of electronic spectra from frozen solutions of complex molecules at lower (liquid helium) temperatures. It was shown that at liquid helium temperatures discrete fluorescence spectra of complex molecules with

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