Structural Changes of Cytochromec552 fromThermus thermophilus Adsorbed on Anionic and Hydrophobic Surfaces Probed by FTIR and 2D-FTIR Spectroscopy

Abstract

The structural changes of cytochrome c(552) bound to anionic and hydrophobic clay surfaces have been investigated by Fourier transform infrared spectroscopy. Binding to the anionic surface of montmorillonite is controlled by electrostatic interactions since addition of electrolyte (0.5 mol L(-1) KCl) causes desorption of more than 2/3 of the protein molecules. Electrostatic binding occurs through the back side of the protein (i.e., remote from the heme site) and is associated only with subtle changes of the secondary structure. In contrast, adsorption to the hydrophobic surface of talc leads to a decrease in alpha-helical structure by ca. 5% and an increase in beta-sheet structure by ca. 6%. These structural changes are attributed to a hydrophobic region on the front surface of cytochrome c(552) close to the partially exposed heme edge. This part on the protein surface is identified as the interaction domain for talc and most likely also serves for binding to the natural reaction partner, a ba(3)-oxidase. Fourier transform infrared spectra of cytochrome c(552) and the clay-cytochrome c(552) complexes have been measured as a function of time following dissolution and suspension in deuterated buffer, respectively. A two-dimensional correlation analysis was applied to these spectra to investigate the dynamics of the structural changes in the protein. For both complexes, adsorption and subsequent unfolding processes in the binding domains are faster than the time resolution of the spectroscopic experiments. Thus, the processes that could be monitored are refolding of peptide segments and side chain rearrangements following the adsorption-induced perturbation of the protein structure and the solvation of the adsorbed protein. In each case, side chain alterations of solvent-exposed tyrosine, aspartate, and glutamate residues were observed. For the cytochrome 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 exchange of amide group protons.

Kinetic and Conformational Insights of Protein Adsorption onto Montmorillonite Revealed Using in Situ ATR-FTIR/2D-COS

“…At higher θ and [BSA] values, a decrease in extended chain/turn structures and side-chain rearrangement seems to occur in order to develop a greater extent of aggregated structures through protein–protein interaction. These results are in contrast to previous 2D-COS analysis of cytochrome c 552 adsorption onto montmorillonite, which highlights the role of β structures and turns in the adsorption process rather than extended chains . Comparing the results between these two studies, however, is challenging with the intrinsic differences between BSA and cytochrome c 552 , which have different properties.…”

“…Comparing the results between these two studies, however, is challenging with the intrinsic differences between BSA and cytochrome c 552 , which have different properties. Cytochrome c 552 possesses a different secondary structure from BSA (cytochrome c 552 has more native β-sheets and fewer α-helices than BSA), likely resulting in different structural implications of adsorption . For the higher concentrations studied here, the proposed pathways from 2D-COS analyses also contrast with existing concepts of BSA unfolding on montmorillonite, which suggests a degradation of helical structures and subsequent conversion into bent structures .…”

Advances in two-dimensional correlation spectroscopy

“…2D IR of cytochrome c 552 from Thermus thermophilus bound to anionic and hydrophilic clay undergoing H/D exchange with the influence of electrostatic interactions was carried out by Lecomte et al [208].…”