A Solution Study on the Local and Global Structure Changes of Cytochrome c:  An Unfolding Process Induced by Urea

Abstract

The local and global structural changes of cytochrome c induced by urea in aqueous solution have been studied using X-ray absorption spectroscopy (XAS) and small-angle X-ray scattering (SAXS). According to the XAS result, both the native (folded) protein and the unfolded protein exhibit the same preedge features taken at Fe K-edge, indicating that the Fe(III) in the heme group of the protein maintains a six-coordinated local structure in both the folded and unfolded states. Furthermore, the discernible differences in the X-ray absorption near-edge structure (XANES) of these two states are attributed to a possible spin transition of the Fe(III) from a low-spin state to a high-spin state during the unfolding process. The perseverance of six-coordination and the spin transition of the iron are reconciled by a proposed ligand exchange, with urea and water molecules replacing the methionine-80 and histidine-18 axial ligands, respectively. The SAXS result reveals a significant morphology change of cytochrome c from a globular shape of a radius of gyration R(g) = 12.8 A of the native protein to an elongated ellipsoid shape of R(g) = 29.7 A for the unfolded protein in the presence of concentrated urea. The extended X-ray absorption fine structure (EXAFS) data unveil the coordination geometries of Fe(III) in both the folded and unfolded state of cytochrome c. An initial spin transition of Fe(III) followed by an axial ligand exchange, accompanied by the change in the global envelope, is proposed for what happened in the protein unfolding process of cytochrome c.

Protein Adsorption into Mesopores: A Combination of Electrostatic Interaction, Counterion Release, and van der Waals Forces

“…Immersion into 8 M urea solutions disrupts the hydrogen bonds which stabilize the cytochrome c's tertiary structure and leads to unfolding of the molecule. 24 Small angle x-ray data 25 of the unfolded cytochrome c at pH 7 reveal a structural transition from the almost spherical conformation of the folded protein (semi-major axis 18 Å, semi-minor axis 18 Å, radius of gyration R g = 12.8 Å) to an eccentric ellipsoid shape (semimajor axis 65 Å, semi-minor axis 9 Å at 8 M urea and R g = 29.7 Å at 10 M urea).…”

“…At pH 4.4, we observe a surface coverage of 1.06 •10 −12 mol/cm 2 and a volume packing density of 5.6%. We obtain the respective values for the unfolded protein using the x-ray scattering data of Hsu et al 25 In 8 M urea solutions, each molecule has a volume of V u = 22 nm 3 and takes up 23.4 nm 2 when adsorbed on a surface. The theoretical monolayer would thus contain 7 • 10 −12 mol/cm 2 .…”

Theoretical Analysis of Fe K-Edge XANES on Iron Pentacarbonyl

“…It can provide coordination numbers and distances between absorbing atom and surrounding atoms within 5 Å. 30 The range above the rising edge up to 40 eV including the pre-edge peaks is named XANES. In the XANES regime, the spectrum features are dominated by the contributions of multiple scattering events with many atoms, which is a measurement of high-order atomic correlation functions.…”

Global and Local Structural Changes of Cytochrome c and Lysozyme Characterized by a Multigroup Unfolding Process

“…Compared to the optical spectroscopies mentioned, small-angle x-ray scattering (SAXS) can be a more direct tool in extracting global structural information of proteins in unfolding (7,8). Recent studies have shown the apparent advantage of combining global and local structural tools in addressing correlations between the local-local structures and local-global structures (9)(10)(11); with this information, a better description of the protein unfolding process as well as a better understanding of intermediate states or partially folded structures can be expected.…”