FLUORESCENCE SPECTRAL PROPERTIES OF TROPONIN-C MUTANT F22W WITH ONE-PHOTON, 2-PHOTON, AND 3-PHOTON EXCITATION

We report the first measurements of protein fluorescence with three-ph oton excitation, using a mutant of troponin C (TnC) that contains a si ngle tryptophan residue F22W. From the emission intensity dependence o n laser power we determine that TnC F22W displays one-, two-, and thre e-photon excitation at 285, 570, and 855 nm, respectively. The emissio n spectra and intensity decays are identical for one-, two-, or three- photon excitation. The steady-state and time 0 anisotropies are distin ct for each mode of excitation, but the correlation times were the sam e, suggesting that three-photon excitation of proteins can be accompli shed without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only ne gative values, suggesting dominant excitation via the (1)L(b) state of tryptophan from 830 to 900 nm.