Structural changes of cytochrome c(552) from Thermus thermophilus adsorbedon anionic and hydrophobic surfaces probed by FTIR and 2D-FTIR spectroscopy

The structural changes of cytochrome C-552 bound to anionic and hydrophobic clay surfaces have been investigated by Fourier transform infrared spectro scopy. Binding to the anionic surface of montmorillonite is controlled by e lectrostatic interactions since addition of electrolyte (0.5 mol L-1 KCl) c auses desorption of more than 2/3 Of the protein molecules. Electrostatic b inding occurs through the back side of the protein (i.e., remote from the h eme site) and is associated only with subtle changes of the secondary struc ture. In contrast, adsorption to the hydrophobic surface of talc leads to a decrease in a-helical structure by ca. 5% and an increase in beta -sheet s tructure by ca. 6%. These structural changes are attributed to a hydrophobi c region on the front surface of cytochrome C-552 close to the partially ex posed heme edge. This part on the protein surface is identified as the inte raction domain for talc and most likely also serves for binding to the natu ral reaction partner, a ba(3)-oxidase. Fourier transform infrared spectra o f cytochrome c(552) and the clay -cylochrome c(552) complexes have been mea sured as a function of time following dissolution and suspension in deutera ted buffer, respectively. A two-dimensional correlation analysis was applie d to these spectra to investigate the dynamics of the structural changes in the protein. For both complexes, adsorption and subsequent unfolding proce sses in the binding domains are faster than the time resolution of the spec troscopic experiments. Thus, the processes that could be monitored are refo lding of peptide segments and side chain rearrangements following the adsor ption-induced perturbation of the protein structure and the solvation of th e adsorbed protein. In each case, side chain alterations of solvent-exposed tyrosine, aspartate, and glutamate residues were observed. For the cytochr ome c(552)-talc complex, these changes are followed by a slow refolding of the peptide chain in the binding domain and, subsequently, a further H/D ex change of amide group protons.