Recognition Of A-Helices In Protein Structures By Iron(III) Complexes Via A Hydrolytic Pathway

Structural information of proteins is of importance for understanding function and mechanisms of proteins in the proteomie times. To date, determination of three-dimensional structures of proteins is heavily dependent on X-ray crystallography and multi-dimensional nuclear magnetic resonance (NMR). The requirement of X-ray crystallography is to obtain a crystal of a protein, whereas NMR is mainly used to determine structures of the proteins with low and middle molecular weights. Therefore, development of assay methods of protein structures is of significance for probing structure-function relationships of a protein. The limited proteolysis has widely been accepted as probe for protein structure. However, the protein hydrolysis catalyzed by natural proteases is only residue-specific, only providing information on the surface exposure. In this work, we found that a dinuclear iron complex [Fe2(DTPB)(μ2-O)(μ2-Ac)Cl] (BF4)2 and a tetranuclear iron (Ⅲ) complex [Fe4(NTB)4(μ2-O)2(μ4-Suc)]6+ are excellent agents that result in protein cleavage via a hydrolytic pathway, and the hudrolysis is not sensitive to residue types in the proteins. The binding interaction between each iron complex and the proteins were firstly probed by UV-visible absorption and CD spectra. For the case of Fe4, the results show that μ2-O bond is cleaved and the α-helical content of proteins is decreased during the binding of Fe4 to proteins. For the case of Fe2, a new species with the strong optical activity is formed by displacing Cl-in Fe2 with some protein residues. The apparent binding constants and apparent binding rate constants of Fe2 or Fe4 to Hb, BSA, Lys, RNase, SOD were obtained. On the other hand, five proteins Hb, BSA, Lys, RNase, SOD with different α-helical content were chosen and their hydrolytic rate and efficiency were also explored. The data clearly demonstrate that natural and partly denatured proteins with high α-helices content (Hb, BSA) are hydrolyzed promoted by Fe4 more easily than those with high β-sheet and disorder structure contents (Lys, RNase, SOD). But, there is not a direct relationship between α-helical content in the proteins and the highest yields of the natural proteins, while in the case of hydrolysis of the partially denatured proteins mediated by Fe2, a linear relationship is clearly observed between the highest yields and the α-helical content in proteins. The products of Hb hydrolytic reaction mediated by Fe2 were examined by LC-MS. Small polypeptide fragments are observed, indicating tat the hydrolysis is not sensitive to residue types in the proteins. Comparison of various parameters that feature the hydrolytic reactions indicates that the Fe2 can be used to obtain the basic information on the secondary structure as a hydrolytic probe of proteins.