Structural and Dynamical Properties of a Partially Unfolded Fe₄S₄ Protein:  Role of the Cofactor in Protein Folding^†

Heteronuclear multidimensional NMR spectroscopy was used to investigate in detail the structural and dynamical properties of a partially unfolded intermediate of the reduced high-potential iron−sulfur protein (HiPIP) from Chromatium vinosum present in 4 M guanidinium chloride solution. After an extensive assignment of ¹⁵N and ¹H resonances, NOE data, proton longitudinal relaxation times, and ³J_HNHα coupling constants as well as ¹⁵N relaxation parameters (T₁, T₂, T_1ρ, and ¹H−¹⁵N NOE) were obtained and used to build a structural model of the intermediate. The Fe₄S₄ cluster of the HiPIP plays a decisive role in determining the resulting structure, which is random in the N-terminal half of the protein and partially organized in the loops between the cysteines bound to the cluster. Consistent with the structural data, the backbone mobility is typical of folded proteins in the regions where there are elements of structure and increases with the structural indetermination.