Time resolved fluorescence of bacteriophage Pfl DNA binding protein and its complex with DNA.

The DNA binding protein of the filamentous bacteriophage Pfl exhibits fluorescence from a single tryptophan residue. The location of the emission maximum at 340 nm ist quite common for proteins, but the single lifetime of 7.8 ns is one of the longest yet reported. Protein fluorescence is quenched more efficiently by Cs+ than by I-; the Trp is located in a partially exposed pocket, in the vicinity of a negative charge. In the native complex of the binding protein with Pfl DNA the fluorescence emission maximum is at 330 nm, indicating a more apolar environment for Trp 14. The native nucleoprotein complex exhibits a similar fluorescence lifetime (6.5 ns) and an approximately equal fluorescence yield, indicating the absence of Trp-DNA stacking. The tryptophan in the complex is virtually inaccessible to ionic quenchers, and thus appears to be buried. Fluorescence depolarisation measurements have been used to examine the rotational mobility of the tryptophan in the protein and in the nucleoprotein complex. In the protein alone a single rotational correlation time (phi) of approximately 19 ns is observed, corresponding to rotation of the entire dimeric molecule; in the native nucleoprotein complex with Pfl DNA, a phi of approximately 500 ns is observed, corresponding to a rigid unit of at least 50 subunits. In neither case does the tryptophan exhibit any detectable flexibility on the subnanosecond time scale.