Spectral fluctuation of a single fluorophore conjugated to a protein molecule

We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal ref lection fluorescence microscope. The most reactive cysteine residue of myos in subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral s hift induced by a change in solvent from aqueous buffer:td methanol in both single-molecule and bulk measurements were similar, indicating that, even at the single molecule level, the fluorescence spectrum is sensitive to mic roenvironmental changes of fluorophores. The time dependence of the fluores cence spectra of fluorophores attached to S1 molecules solely showed a fluc tuation with time over a time scale of seconds. Because the fluorescence sp ectra of the same fluorophores directly conjugated to a glass surface remai ned constant, the spectral fluctuation observed for the fluorophores attach ed to S1 is most likely due to slow spontaneous conformational changes in t he S1 molecule. Thus, single-molecule fluorescence spectroscopy appears to be a powerful tool to study the dynamic behavior of single biomolecules.