Junxia Lu

Effects of charged amino‐acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b5 mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045±0.009 nm and 0.088±0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X‐ray and solution structures of wild‐type cytochrome b5. The binding of different surface mutants of cytochrome b5 with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b5 surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c‐cyt b5 complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b5, do occur in the association between cytochrome b5 and cytochrome c.

Structure of the F58W mutant of cytochrome b5: the mutation leads to multiple conformations and weakens stacking interactions

Phe58 of cytochrome b5 is involved in stacking interactions with heme and the axial ligand His63. To elucidate the contribution of the stacking interactions to protein stability, the crystal structures of the F58Y and F58W mutants were determined at high resolution. The structure of the F58Y mutant is basically the same as that of the wild-type protein. However, the mutation from Phe58 to Trp58 leads to difficulty in growing single crystals and results in a space-group change; the six molecules in the asymmetric unit form two groups that are related by a non-crystallographic twofold axis. The structure of F58W was determined using molecular replacement by making use of the non-crystallographic symmetry. The F58W mutation gives rise to multiple conformations of six side chains, a peptide linking two of the six residues and the extended propionic acid of the heme. The six molecules in the asymmetric unit of the F58W mutant structure are grouped into two types based on their conformations and one of the six molecules exhibits dual conformations. The stacking interactions are weakened owing to the increase/decrease of the angles between the indole ring of Trp58 and the His63/heme rings, which accounts for the lower stability of F58W compared with the wild-type protein.

Study on the DME-cytochromeb 5 and its mutants at site of F58

Phenylalanine-58 is one of the conservative residues in the hydrophobic pocket of Cytb5, which forms aromatic stacking with the hemeb. Previous study showed that both the stacking and the property of the aromatic residue affect hydrophobicity of the heme pocket, leading to change of protein’s property. In order to further reveal the essence we esterify the heme propionate of Cytb5, F58Y.and F58W, and eliminate the hydrogen bond between heme propionate and Ser64 in examining the effect of hydrogen net on the π-π interaction. In this paper thermal denaturation of DME-Cytb5 and its F58Y.and F58W mutants has been studied by UV-visible and CD spectra. The heme transfer reactions between these proteins and apo-myoglobin have been studied as well. The results demonstrate that esterification did not destroy the aromatic stacking; however, it affects the stability of the proteins due to different volumes, hydrophobicities and hydrogen bonds forming ability of these substituents.

Preparation and characterization of some surface negatively charged residue mutants of cytochrome b5

Site-directed mutagenesis was used to obtain seven variants of tryptic fragment of bovine liver cytochrome bm5 (cyt b5), in which the negatively charged residues around the heme exposed edge of cyt b5 were replaced by hydrophobic amino acid alanine. Double-site mutants, triple-site mutants and even quadruple-site mutants were obtained. DNA sequencing and molecular weight measurements of the mutant proteins both confirmed that these site-directed mutagenesises were successfully performed. Spectroelectrochemistry of these mutant proteins revealed that the apparent redox potentials of these mutant proteins caused a positive shift of 2–10 mV. The global structure of these mutant proteins did not show much difference from that of the wild type cyt b5, providing a solid base for the further study on the roles of the proteins’ surface charges.