Effects of Denaturant and Pressure on the Intrinsic Fluorescence of Titin

The intrinsic fluorescence of titin was analyzed in this present study. The average fluorescence wavelength of titin was 17 nm shorter than that of tryptophan in water, indicating that tryptophans in titin are not completely exposed to solvent. Guanidine hydrochloride (GdmCl) induced a red spectral shift of 17 nm, a decrease of total intensity of 33%, an increase of spectral bandwidth, and a decrease of anisotropy of 59%. The GdmCl concentration dependence of these fluorescence parameters indicated a two-state transition phenomenon with a transition midpoint at 2.6 M. The free energy change associated with this transition is in the range of 3-3.5 kcal/mol/domain, assuming that titin domains are unfolded independently. The anisotropy value also decreased 69% upon application of hydrostatic pressure up to 2.4 kbar, similar to the situation of GdmCl denaturation. However, the spectral red shift was 3.7-nm at a pressure of 2.4 kbar, indicating that the pressure-altered conformation of titin at 2.4 kbar is distinct to the GdmCl-denatured conformation. Pressure above 2 kbar apparently generated a flexible hydrophobic pocket for tryptophan residues of titin.