THE APPLICATION OF STYRYL AND SQUARAINE DYES FOR DETERMINATION OF CONFORMATION CHANGES IN PROTEIN MOLECULES

Abstract

It is known, that the application of Förster Resonance Energy Transfer (FRET) between two dyes absorbing in different spectrum regions allows shifting of the excitation wavelength into the blue region. We investigated spectral properties and FRET between styryl (St) and squaraine (Sq) dyes to evaluate the possibility of using such FRET-pair to determine conformational changes in proteins. Spectral properties of St and Sq in polar (phosphate buffer), nonpolar (chloroform) solvents and in non-covalent complexes with BSA (bovine serum albumin) and HSA (human serum albumin) were studied. The effect of changes in protein conformation induced by urea solutions of various concentrations on the spectral properties of dyes and energy transfer efficiency was also studied. It was found that the polarity of the medium affects the fluorescent properties of dyes in different ways: styryl dye has a positive solvatofluorochromism, while the squaraine dye has the negative one. Both dyes show negative solvatochromism, and their quantum yields substantially increase in a less polar environment. Despite the spectral differences of the St-BSA and St-HSA complexes, their quantum yields are the same, and in case of squaraine dye the quantum yield for Sq-HSA is almost twice higher that for Sq-BSA. When the conformation of protein changes, the dyes go into the aqueous phase, as a result the intensity of their fluorescence decreases almost to the level of fluorescence of the free dye in phosphate buffer. There are two bands corresponding to the donor and acceptor in the fluorescence spectra of the double complexes St-BSA-Sq and St-HSA-Sq. The pair of styryl-squaraine dyes is differently sensitive to the conformation changes in these albumins caused by urea: the FRET efficiency for HSA changes by 11.5 times, while for BSA only by 2.1 times. Thus, the St–Sq pair has the ability to detect a change in the conformation of proteins, but the efficiency of the pair depends on the properties of the protein under study.

Keywords

• fret, стириловий барвник, сквараїновий барвник, альбуміни, зміни конформації