Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the a-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the Nterminus (b-domain) and one in the C-terminus (a-domain). The overall fold of the a-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the b-domain exhibits similar internal motion to the a-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.

We found that cyt c Y67H and Y67R variants represent a state which resembles the conformational intermediate state in cyt c with high peroxidase activity; and also the hydrogen bond network around Tyr67 is associated with the conformational transition of cyt c; these suggest that the hydrogen bond network around Tyr67 is essential in maintaining the cyt c functioning not only as an electron transfer protein but also probably as a trigger in apoptosis.

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.

The β-amyloid peptide (Aβ) aggregation in the brain, known as amyloid plaques, is a hallmark of Alzheimer's disease (AD). The aberrant interaction of Cu(2+) ion with Aβ potentiates AD by inducing Aβ aggregation and generating neurotoxic reactive oxygen species (ROS). In this study, the biosynthesized recombinant Aβ(1-40) was, for the first time, used to investigate the mechanism for heme to prevent Aβ(1-40) aggregation and its cytotoxicity. Cell viability studies of SH-SY5Y cells and rat primary hippocampal neurons showed that exogenous heme can protect the cells by reducing cytotoxicity in the presence of Cu(2+) and/or Aβ(1-40). UV-vis spectroscopy, circular dichroism spectroscopy, and differential pulse voltammetry were applied to examine the interaction between heme and Aβ(1-40). It was proven that a heme-Aβ(1-40) complex is formed and can stabilize the α-helix structure of Aβ(1-40) to inhibit Aβ(1-40) aggregation. The heme-Aβ(1-40) complex possesses peroxidase activity and it may catalyze the decomposition of H(2)O(2), reduce the generation of ROS downstream, and ultimately protect the cells. These results indicated that exogenous heme is able to alleviate the cytotoxicity induced by Aβ(1-40) and Cu(2+). This information may be a foundation to develop a potential strategy to treat AD.

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.