Fluorescence and excitation Escherichia coli RecA protein spectra analyzedseparately for tyrosine and tryptophan residues

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 c oli RecA protein and its complexes with Mg2+, ATP gamma S or ADP, and singl e-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 T residues in the C-terminal domain was shown to be quenched by energy transf er 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 a nd Tyr constituents revealed that a relative spatial location of the C-term inal and the major domains in RecA monomers was different for their complex es with either ATP gamma S or ADP, whereas this location did not change upo n additional interaction of these complexes with ssDNA. Homogeneous (that i s, independent of EX wavelength) and nonhomogeneous (dependent on EX wavele ngth) types of Tyr and Trp fluorescence quenching were analyzed for RecA an d its complexes with nucleotide cofactors and ssDNA The former was expected to result from singlet-singlet energy transfer from these residues to aden ine 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 o f Trp and Tyr residues to the adenine. In this case, for correct calculatio n of the overlap integral, Trp and Tyr donor emission spectre were substitu ted by the spectra function of convolution of emission and excitation spect ra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-te rminal domain. (C) 2000 Academic Press.