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Proceedings Paper

Fluorimetric characterization of tryptophan residues in Escherichia coli single-stranded DNA-binding (SSB) protein and its poly(dT) complex
Author(s): Jose Ramon Casas-Finet
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Paper Abstract

The individual contribution of the 4 tryptophan residues (at positions 40, 54, 88, and 135) of E.coli SSB to its fluorescence emission was elucidated by deconvolution of the spectral envelope of wild type (wt), singly (-W40F, -W54F, -W88F, -W135F), and doubly (-W88F+W135F) point-mutated SSBs. Upon excitation at 297 nm, Trp 40, Trp 54, Trp 88, and Trp 135 peaked at 342, 347, 344, and 352 nm, respectively, and accounted for 22, 43, 24, and 11% of the SSB integrated emission. Extensive Tyr to Trp energy transfer at the singlet level was detected. Upon binding poly(dT), the emission spectra of all SSBs containing Trp 135 was red-shifted by 3 to 5 nm and their linewidth increased by ca. 4%, whereas for SSBs lacking Trp 135 more pronounced broadening without spectral shift was observed. In poly(dT) complexes, the limiting quenching (Qmax) at saturation (relative to the free protein) for the various SSB mutants was 93, 90, 89, 88, 85, and 79% for SSB-W88F+W135F, SSB-W135F, wt SSB, SSB-W88F, SSB-W40F, and SSB-W54F, respectively. From these Qmax values and the contribution of individual Trp residues to the overall emission of free SSB, the calculated individual Trp Qmax were 92, 95, 81, and 59% for residues 40, 54, 88, and 135, respectively. Trp 40 and Trp 54, shown earlier to be the only two Trp residues involved in stacking interactions with nucleic acid bases in SSB/polynucleotide complexes, exhibit almost complete quenching. In wt SSB/poly(dT) complex, Trp 40, 54, 88, and 135 account for 14, 18, 33, and 36% of the total emission. The dominance of the red-emitting Trp 135 in the fluorescence of SSB/poly(dT) complexes accounts for the spectral red-shift in systems containing this residue. Global analysis of fluorescence lifetime experiments in the frequency domain showed that Trp 54 has the longest average lifetime (8.0 ns). The long lifetime of Trp 54 appears to be due to its particular microenvironment, since that average lifetime for all individual Trp were in the 2 to 4 ns range in 8 M urea.

Paper Details

Date Published: 17 August 1994
PDF: 7 pages
Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); doi: 10.1117/12.182709
Show Author Affiliations
Jose Ramon Casas-Finet, National Cancer Institute (United States)


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

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