Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 376, Issue 1, Pages 124-140Publisher
ACADEMIC PRESS INC
DOI: 10.1006/abbi.2000.1698
Keywords
tyrosine and tryptophan RecA protein; emission and excitation spectra; homogeneous and nonhomogeneous fluorescence quenching; singlet-singlet energy transfer; RecA protein structure-function relationships
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The method for separation of emission (EM) and excitation (EX) spectra of a protein into EM and EX spectra of its tyrosine (Tyr) and tryptophan (Trp) residues was described, The method was applied to analysis of Escherichia coli RecA protein and its complexes with Mg2+, ATP gamma S or ADP, and single-stranded DNA (ssDNA), RecA consists of a C-terminal domain containing two Trp and two Tyr residues, a major domain with five Tyr residues, and an N-terminal domain without these residues (R. M. Story, I. T. Weber, and T. A. Steitz (1992) Nature (London) 355, 374-376), Because the fluorescence of Tresidues in the C-terminal domain was shown to be quenched by energy transfer to Trp residues, Trp and Tyr fluorescence of RecA was provided by the C-terminal and the major domains, respectively. Spectral analysis of Trp yr and Tyr constituents revealed that a relative spatial location of the C-terminal and the major domains in RecA monomers was different for their complexes with either ATP gamma S or ADP, whereas this location did not change upon additional interaction of these complexes with ssDNA. Homogeneous (that is, independent of EX wavelength) and nonhomogeneous (dependent on EX wavelength) types of Tyr and Trp fluorescence quenching were analyzed for RecA and its complexes with nucleotide cofactors and ssDNA The former was expected to result from singlet-singlet energy transfer from these residues to adenine of ATP gamma S or ADP, By analogy, the latter was suggested to proceed through energy transfer from high vibrational levels of the excited state of Trp and Tyr residues to the adenine. In this case, for correct calculation of the overlap integral, Trp and Tyr donor emission spectre were substituted by the spectra function of convolution of emission and excitation spectra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-terminal domain. (C) 2000 Academic Press.
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