The Combined Use of Fluorescence Spectroscopy and X-Ray Crystallography Greatly Contributes to Elucidating Structure and Dynamics of Proteins

Intrinsic fluorescence is a powerful tool for investigating the structure, dynamics, and folding-unfolding of proteins (Lakowicz, 1999; Eftink, 1991, 1998; Demchenko, 1986; Burshtein, 1976). This is due to high sensitivity of various parameters of the fluorescence of tryptophan residues (spectrum position, quantum yield, anisotropy, etc.) to their microenvironment and to the peculiarities of their location in protein macromolecules. The dependence of intrinsic protein fluorescence on the unique location of tryptophan and tyrosine residues was elaborated by the study of intrinsic fluorescence of model compounds and proteins in different structural states. The combined analysis of the characteristics of protein intrinsic fluorescence and protein three-dimensional structure with the peculiarities of the location of tryptophan residues in protein structure was performed for the first time on azurin (Turoverov et al., 1985). The dependence of the recorded fluorescence characteristics of tryptophan residues on the peculiarities of their microenvironment has been further studied on other proteins (Turoverov and Kuznetsova, 1986; Agekyan et al., 1988; Kuznetsova et al., 1996, 1999, 2000, Stepanenko, 2004, Staiano et al., in press). Carrying out these studies on recombinant proteins with altered tryptophan residues or other amino acid residues in the vicinity of indol residues essentially increases the experimental basis of these works (Martensson et al., 1995; Gopalan et al., 1997; Atkins et al., 1991; Doyle et al., 2001). Since detailed spatial structures of many proteins (co-ordinates of separate atoms in protein structures) determined by X-ray analysis are available from Protein Data Bank (Bernstein et al., 1977) this prompts the idea to fulfill the combined analysis of the characteristics of protein intrinsic fluorescence and their 3-D structures in order to elucidate the peculiarities of protein microenvironments such as protein dynamics and protein folding-unfolding processes