ИСТИНА

Fluorescent proteins (FPs) are a powerful tool for imaging of single molecules, intact organelles, live cells, and whole organisms. Fluorescence microscopy has become an invaluable approach in the fields of biochemistry, biotechnology, cell and developmental biology. Recent developments of FPs with light-modulated spectral properties, far-red FPs and FPs with a large Stokes shift have enabled the imaging of fixed and live cells using super-resolution imaging techniques and the visualization of cellular processes in animals. Because the proper fold of -barrels underlies the acquisition of FP functions (i.e., the ability to fluoresce), the study of FP folding appears to be important. The folding of proteins with high content of -structure is per se an important fundamental problem to which a number of studies are dedicated. At the same time, in none of these works the complex shape of absorption spectrum of FP in buffered solutions [1] and its alterations with the change of solution composition were taken into account. Even in the study of an unfolding–refolding process of yellow FP (EYFP), whose absorption is quite sensitive to the presence of anions, such as halides, nitrates, and thiosulfate [2], the chromophore fluorescence intensity was not corrected to the change of absorption at the excitation wavelength [3]. However, it must be done not only for EYFP but for any FP. Careful examination of the unfolding-refolding of green fluorescent protein sfGFP induced by guanidine thiocyanate (GTC) and guanidine hydrochloride (GdnHCl) showed that these denaturing agents induce pronounced alterations in visible absorption spectrum of sfGFP, especially in the range of 0–0.7 M GTC and 0–2.5 M GdnHCl. In these ranges of denaturants concentration, the visible absorption spectra of sfGFP demonstrated a sharp decrease in the absorption band at 485 nm, which corresponds to the anionic form of the chromophore, with a concomitant increase in the absorption band at 390 nm, which corresponds to the neutral chromophore. It is important that in this range of denaturants concentrations the changes of sfGFP chromophore and tryptophan fluorescence are observed that erroneously can be mixed up with appearance of an intermediate state. A joint analysis of the chromophore fluorescence corrected on the changes of absorption at excitation wavelength, fluorescence anisotropy and the parameter A, and the change of elution volume of sfGFP has shown that the protein structure is preserved in this conditions. Denaturing agents in higher concentrations induce a disruption of the sfGFP structure, which is manifested in simultaneous changes of all of the examined parameters. Tryptophan and chromophore fluorescence of sfGFP was studied in solutions with small concentrations of chemical denaturants (GTC and GdnHCl) or with different salt concentrations (NaCl, sodium thiocyanate). It was shown that dramatic changes of spectral characteristics of sfGFP in the presence of tested agents are caused by binding of negatively charged ions, such as SCN– and Cl–, in the vicinity of the chromophore. Presumably, the SCN– and Cl– anions have different binding sites at the sfGFP protein.