Estudos de fluorescência da proteína recombinante cofilina-1 humana

Abstract

Intrinsic fluorophore groups natively present in proteins, such as tryptophan residues, and extrinsic fluorescent probes, such as 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS), can be employed in the monitoring of conformational changes caused by the interaction of small molecules or metallic ions with proteins structure by the fluorescence quenching phenomenon. For this purpose, the human cofilin-1, a potentially involved in Parkinson’s disease (PD) protein, was recombinantly produced in E. coli, purified and submitted to structural investigation in view of its possible interaction with metallic ions of biological interest, such as iron (Fe3+), zinc (Zn2+) and copper (Cu2+), and aluminum (Al3+), a non-physiological metal ion, by fluorometric titration. Where, in PBS, Tris-HCl, TES and HEPES buffers at 20 mM and pH 7,4, the wavelength of maximum fluorescence emission (λmax) of cofilin-1 remained in the range of 329 and 333 nm and the addition of 50-15 mM NaCl, as well of the 1 mM EDTA chelator to the working buffer, 20 mM HEPES pH 7,4, did not influence the protein emission profile. Furthermore, intrinsic fluorescence assays with Zn2+ and Al3+ ions suggested a rapid metal-protein interaction, while extrinsic fluorescence experiments employing bis-ANS (KD = 3,911 µM, R2 = 0,9956) indicated a stronger interaction of Fe3+ (KD = 16,80 μM, R² = 0,9844) and Cu2+ with cofilin-1. The quenching profiles obtained for both Cu2+ and Zn2+ ions suggest a slow interaction kinetics, while the profile obtained for the Al3+ ion indicated a possible transient interaction with the native human cofilin-1 protein. These results, although preliminary, are promising and may eventually serve as a basis for future fluorescence assays using human cofilin-1 protein in its oxidized form.