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Proceedings Paper

Steady-state and time-resolved phosphorescence of 5-hydroxy-L-tryptophan lambda cI repressor bound to DNA
Author(s): Aaron K. Sato; Eric R. Bitten; Drew Lambert; Kenneth W. Rousslang
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Paper 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.

Paper Details

Date Published: 17 August 1994
PDF: 10 pages
Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); doi: 10.1117/12.182743
Show Author Affiliations
Aaron K. Sato, Univ. of Puget Sound (United States)
Eric R. Bitten, Univ. of Puget Sound (United States)
Drew Lambert, Univ. of Puget Sound (United States)
Kenneth W. Rousslang, Univ. of Puget Sound (United States)


Published in SPIE Proceedings Vol. 2137:
Time-Resolved Laser Spectroscopy in Biochemistry IV
Joseph R. Lakowicz, Editor(s)

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