Steady-state and time-resolved phosphorescence of 5-hydroxy-L-tryptophan lambda cI repressor bound to DNA

摘要

The spectral overlap of tryptophan containing proteins and DNA has limited the use of luminescence methods to investigate protein-nucleic acid interactions. However, the steady-state and time-dependent phosphorescence of wild-type and 5-hydroxy-L- tryptophan (5-OHTrp) $lambda cI repressor are spectroscopically distinct such that we can selectively excite the 5-OHTrp-$lambda cI in the presence of DNA, or even in the presence of a 15-fold molar excess of N-acetyl-tryptophanamide (NATrpA). The phosphorescence of wild-type $lambda cI is red-shifted by 3 nm relative to NATrpA, characteristic of buried tryptophan, and the phosphorescence of the spectrally enhanced protein (SEP), 5-OHTrp-$lambda cI repressor is also red shifted relative to the model, 5-OHTrp, providing spectroscopic evidence that the modified repressor is structurally equivalent to the native repressor. Although the phosphorescence decays of NATrpA and 5-OHTrp are simple exponentials, the decay of either wild-type or 5-OHTrp-$lambda cI repressors requires three exponentials whose fractional contributions to the phosphorescence are similar. Since the 5-OHTrp phosphorescence can be excited without interference from tryptophan or DNA, we measured the phosphorescence of the SEP/DNA complex. The emission characteristics of SEP alone and the SEP/DNA complex are indistinguishable, showing that during binding, the C-terminal domain of the protein, believed to be involved in protein dimer stabilization, is structurally conserved in the vicinity of the three modified trytptophans.