ИСТИНА

Fluorescent proteins (FPs) are widely used in diverse biological applications. The study of FP folding is of high importance for understanding of their spectral properties. Superfolder variant of the green fluorescent protein (sfGFP) became a favorite probe for examination of the unfolding-refolding processes of FPs owing to its reversible unfolding in comparison with other FPs. sfGFP is able to fold correctly even being fused with poorly folding polypeptides. Previously we have shown [1] that spectral characteristics of sfGFP are sensitive to small concentrations of ionic denaturant, such as guanidine thiocyanate (GTC). At the same time it was revealed by circular dichroism and gel filtration that the sfGFP structure undergoes only subtle changes in these conditions. Thus, firstly, the fluorescence data obtained in the study of unfolding – refolding processes of FPs induced by ionic denaturants should be analyzed with care, and, secondly, it is interesting to define the reasons of the essential changes of spectral characteristics of sfGFP in the presence of small amount of denaturant. Here, we studied the impact on spectral characteristics and structure of sfGFP of ionic denaturant guanidine hydrochloride (GdnHCl) and GTC, and salts, such as sodium chloride (NaCl) and sodium thiocyanate (NaSCN). It was found that complex pattern of the changes of fluorescent characteristics of sfGFP in the presence of ionic denaturants and salts is concerned with the existence of two types of sfGFP molecules that contain neutral or anionic chromophore and with the shift of the content of these molecules. The redistribution of sfGFP molecules with neutral and anionic chromophore is most pronounced in the presence of GTC and NaSCN, less profound in NaCl, and neglected in GdnHCl. Non-equivalent response sfGFP to studied agents argues for their specific influence. The inhibition of the anionic chromophore of sfGFP in the presence of studied agents indicates on the binding of negatively charged ions of Cl- and SCN-. Moreover, the behavior of spectral characteristics of sfGFP in the presence of NaCl indicated the occurrence of the third type of sfGFP molecule which is likely the protein containing the anionic chromophore with bound Cl- ion. On the basis of absorption data we calculated the molar absorption spectra of sfGFP with neutral, anionic chromophore and anionic chromophore with bound anion in its vicinity by non-linear regression. Our results show that when ionic denaturants are used in the studies of fluorescent protein folding their effect on fluorophore charge state should be taken into account.