Ru(III)(NH(,3))(,5)His complexes are found to be useful as probes of protein structure, because of the following unique physical properties: (1) The ruthenium moiety is useful for the assignment of His proton resonances in the NMR spectra of proteins. Ru(III)(NH(,3))(,5)His is paramagnetic and low spin d('5) in the ground state. The presence of ruthenium affects the frequencies and/or linewidths of the labeled His ('1)H resonances. The ability to assign ('1)H resonances was tested with RNase A since all four CE1 resonances and one CD2 resonance of RNase A can be resolved at low pH. A Ru(III)(NH(,3))(,5)His(.)RNase A derivative, labeled at His 105, was identified by two-dimensional paper electrophoresis and amino acid analysis. Two ('1)H resonances are substantially shifted and/or broadened in the NMR spectrum of this derivative. The assignment of the CD2 and a CE1 resonance to His 105 agrees with previous studies. (2) The Ru(III)(NH(,3))(,5)His complex is useful for the measurement of interresidue distances by fluorescence energy transfer. The complex exhibits ligand-to-metal charge transfer bands that overlap the emission of intrinsic protein fluorophores. Lysozyme and (alpha)-lytic protease were chosen as model systems since each protein has a single His residue, a limited number of Trp donors, and a known x-ray structure. The ruthenium moiety quenches the protein fluorescence in a manner predicted from the x-ray distances and the energy transfer mechanism. Solution-phase structural information is obtained on calmodulin, a ubiquitous Ca('2+)-binding protein. A range of distances from the two tyrosines to Ru(III)(NH(,3))(,5)His(.)107 is obtained by fluorescence energy transfer experiments. The distances calculated are in good agreement with those predicted from residues in analogous positions in a structurally homologous protein, troponin C. These results support the hypothesis that Ca('2+)-binding proteins are structurally homologous (R. H. Kretsinger, CRC Critical Reviews in Biochemistry, 8, 119-174, 1980).
