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

Aaron K. Sato; Eric R. Bitten; Drew Lambert; Kenneth W. Rousslang

doi:10.1117/12.182743

17 August 1994 Steady-state and time-resolved phosphorescence of 5-hydroxy-L-tryptophan lambda cI repressor bound to DNA

Aaron K. Sato, Eric R. Bitten, Drew Lambert, Kenneth W. Rousslang

Author Affiliations +

Proceedings Volume 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV; (1994) https://doi.org/10.1117/12.182743
Event: OE/LASE '94, 1994, Los Angeles, CA, United States

Abstract

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-O1-ITrp) X ci repressor are spectroscopically distinct such that we can selectively excite the 5-OHTrp-?. 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 X 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-X 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-X ci repressors requires three exponentials whose fractional contributions to the phosphorescence are similar. Since the 5-OFITrp phosphorescence can be excited without interference from tryptophan or DNA, we measured the phosphorescence of the SEP/DNA complex. Th 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.

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Aaron K. Sato, Eric R. Bitten, Drew Lambert, and Kenneth W. Rousslang "Steady-state and time-resolved phosphorescence of 5-hydroxy-L-tryptophan lambda cI repressor bound to DNA", Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); https://doi.org/10.1117/12.182743

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