Photophysics of the Tryptophan Residue in RNaseTl and RNase Tl-2′GMP Complex
The photophysics of a single tryptophan residue in RNaseTl was studied by time correlated single photon counting. A pH dependent transition in the fluorescence decay was observed, centered at pH 6.5-7.0. At pH values above this transition, tryptophan fluorescence decay can be described in terms of a double exponential, with τ1 3.9 ns and τ2 1.6 ns. Fluorescence was quenched with acrylamide, which quenches the short lifetime component five times faster than it does the long lifetime component. These results were interpreted with a two state model. At pH′s below the transition, the fluorescence decay of tryptophan in RNaseTl is a single exponential with τ1 4.0 ns. Binding of 2′GMP, an enzyme inhibitor, to RNaseTl caused a change in average fluorescence lifetime and in the conformation distribution of the tryptophan residue in the protein. The fluorescence anisotropy of RNaseTl showed an apparent hydration volume at pH 7.4 which corresponds to a single layer of water molecules bound on the surface of the protein, whereas at pH 5.5, the fluorescence anisotropy did not follow the normal Stokes-Einstein hydrodynamic law.