Structural Perturbation of Superfolder GFP in the Presence of Guanidine Thiocyanate

Figure 1

GTC-induced folding-unfolding of sfGFP recorded by the changes of fluorescence intensity at 320 nm (a) and at 365 nm (b), parameter A (c), fluorescence anisotropy at 365 nm (d), and chromophore fluorescence intensity excited at 365 nm (e) and 470 nm (f). Unfolding and refolding are presented by circles and squares, respectively. Measurements were performed after 1 h (closed circles) and 24 h (open circles and squares), 45 h (triangles), 69 h (reversed triangles), and 94 h (gray crosses) incubation of native or denatured protein in the presence of corresponding denaturant concentration. Data presented in (e) and (f) were corrected to take into account the change of optical density at the wavelength of excitation (g and h). The conformational changes of sfGFP are further characterized by the changes of absorbance at 390 (squares, i) and 490 nm (circles, i). Insert to panel (i): The visible fluorescence spectra of sfGFP in the presence of increasing GTC concentration (shown by arrows). The change of sfGFP hydrodynamic dimensions during GTC-induced denaturation was studied by gel filtration method (j). The position of elution peaks of compact and denatured molecules (gray circles and squares) and the change of averaged elution volume of sfGFP (black triangles) are shown.