Probing the Molecular Mechanism of Human Soluble Guanylate Cyclase Activation by NO in vitro and in vivo.

Soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. NO binds to the heme of sGC to catalyze the synthesis of the second messenger cGMP, which plays a critical role in several physiological processes. However, the molecular mechanism for sGC to mediate the NO signaling remains unclear.

The molecular mechanism of heme loss from oxidized soluble guanylate cyclase induced by conformational change.

Heme oxidation and loss of soluble guanylate cyclase (sGC) is thought to be an important contributor to the development of cardiovascular diseases. Nevertheless, it remains unknown why the heme loses readily in oxidized sGC. In the current study, the conformational change of sGC upon heme oxidation by ODQ was studied based on the fluorescence resonance energy transfer (FRET) between the heme and a fluorophore fluorescein arsenical helix binder (FlAsH-EDT2) labeled at different domains of sGC β1.

Structural basis for cytochrome c Y67H mutant to function as a peroxidase.

The catalytic activity of cytochrome c (cyt c) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt c Y67H variant with high peroxidase activity, which is almost similar to that of its native form.

Exploring the differences between mouse mAβ(1-42) and human hAβ(1-42) for Alzheimer's disease related properties and neuronal cytotoxicity.

The differences between mouse mAβ(1-42) and human hAβ(1-42), explored using CD and fluorescence spectroscopy, transmission electron microscopy, ROS fluorescent assay, and neuronal cell viability, revealed that mAβ(1-42) as a three-site mutant (R5G, Y10F and H13R) of hAβ(1-42) altered the metal (copper and zinc) binding sites, reduced the proneness to form β-sheet structures and aggregated fibrils, alleviated the generation of ROS, and decreased the cytotoxicity, in contrast to hAβ(1-42).

Generation of novel functional metalloproteins via hybrids of cytochrome c and peroxidase.

The continued interest in protein engineering has led to intense efforts in developing novel stable enzymes, which could not only give boost to industrial and biomedical applications, but also enhance our understanding of the structure-function relationships of proteins. We present here the generation of three hybrid proteins of cytochrome c (cyt c) and peroxidase via structure-based rational mutagenesis of cyt c. Several residues (positions 67, 70, 71 and 80) in the distal heme region of cyt c were mutated to the highly conserved amino acids in the heme pocket of peroxidases.

Functional conversion of nickel-containing metalloproteins via molecular design: from a truncated acetyl-coenzyme A synthase to a nickel superoxide dismutase.

Truncated acetyl-coenzyme A synthase (ACS) was successfully converted into functional nickel superoxide dismutase (Ni-SOD) by molecular design and the designed metalloproteins possess new spectroscopic, structural, and electrochemical characteristics required for catalyzing O(2)(˙-) disproportionation, and exhibit impressive Ni-SOD activity.

The molecular mechanism for human metallothionein-3 to protect against the neuronal cytotoxicity of Aβ(1-42) with Cu ions.

Aggregation and cytotoxicity of Aβ with redox-active metals in neuronal cells have been implicated in the progression of Alzheimer disease. Human metallothionein (MT) 3 is highly expressed in the normal human brain and is downregulated in Alzheimer disease. Zn(7)MT3 can protect against the neuronal toxicity of Aβ by preventing copper-mediated Aβ aggregation, abolishing the production of reactive oxygen species (ROS) and the related cellular toxicity.

Structural and functional insights into the heme-binding domain of the human soluble guanylate cyclase α2 subunit and heterodimeric α2β1.

Soluble guanylate cyclase (sGC) mediates NO signaling for a wide range of physiological effects in the cardiovascular system and the central nervous system. The α1β1 isoform is ubiquitously distributed in cytosolic fractions of tissues, whereas α2β1 is mainly found in the brain. The major occurrence and the unique characteristic of human sGC α2β1 indicate a special role in the mediation of neuronal communication.

Conformational toggling of yeast iso-1-cytochrome C in the oxidized and reduced states.

To convert cyt c into a peroxidase-like metalloenzyme, the P71H mutant was designed to introduce a distal histidine. Unexpectedly, its peroxidase activity was found even lower than that of the native, and that the axial ligation of heme iron was changed to His71/His18 in the oxidized state, while to Met80/His18 in the reduced state, characterized by UV-visible, circular dichroism, and resonance Raman spectroscopy. To further probe the functional importance of Pro71 in oxidation state dependent conformational changes occurred in cyt c, the solution structures of P71H mutant in both oxidation states were determined.

A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase.

Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible.

The mechanism for heme to prevent Aβ(1-40) aggregation and its cytotoxicity.

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.

Efficient expression and purification of methyltransferase in acetyl-coenzyme a synthesis pathway of the human pathogen Clostridium difficile.

The Wood-Ljungdahl pathway is responsible for acetyl-CoA biosynthesis and used as a major mean of generating energy for growth in some anaerobic microbes. Series of genes, from the anaerobic human pathogen Clostridium difficile, have been identified that show striking similarity to the genes involved in this pathway including methyltetrahydrofolate- and corrinoid-dependent methyltransferase. This methyltransferase plays a central role in this pathway that transfers the methyl group from methyltetrahydrofolate to a cob(I)amide center in the corrinoid iron-sulfur protein.

Probing the role of the bridging C509 between the [Fe4S4] cubane and the [Ni(p)Ni(d)] centre in the A-cluster of acetyl-coenzyme A synthase.

The A-cluster of acetyl-coenzyme A synthase consists of an [Fe(4)S(4)] cubane bridged to a [Ni(p)Ni(d)] centre via C509 cysteinate. The bridging cysteinate, which could be substituted by histidine imidazole, mediates "communication" between the [Fe(4)S(4)] cubane and the [Ni(p)Ni(d)] centre during the synthesis of acetyl-coenzyme A.

Structural and functional insights into polymorphic enzymes of cytochrome P450 2C8.

The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of drugs and exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver. Nearly all previous works about polymorphic variants of CYP2C8 were focused on unpurified proteins, either cells or human liver microsomes; therefore their structure-function relationships were unclear.

Neuronal growth-inhibitory factor (metallothionein-3): structure-function relationships.

Neuronal growth-inhibitory factor (GIF), also named metallothionein-3, inhibits the outgrowth of neuronal cells. Recent studies on the structure of human GIF, carried out using NMR and molecular dynamics simulation techniques, have been summarized. By studying a series of protein-engineered mutants of GIF, we showed that the bioactivity of GIF is modulated by multiple factors, including the unique TCPCP motif-induced characteristic conformation, the solvent accessibility and dynamics of the metal-thiolate cluster, and the domain-domain interactions.

The Delta33-35 Mutant alpha-Domain Containing beta-Domain-Like M(3)S(9) Cluster Exhibits the Function of alpha-Domain with M(4)S(11) Cluster in Human Growth Inhibitory Factor.

Neuronal growth inhibitory factor (GIF), also known as metallothionein (metallothionein-3), impairs the survival and neurite formation of cultured neurons. It is known that the alpha-beta domain-domain interaction of hGIF is crucial to the neuron growth inhibitory bioactivity although the exact mechanism is not clear. Herein, the beta(MT3)-beta(MT3) mutant and the hGIF-truncated Delta33-35 mutant were constructed, and their biochemical properties were characterized by pH titration, EDTA, and DTNB reactions.

Distinct mechanisms for the pro-apoptotic conformational transition and alkaline transition in cytochrome c.

We demonstrate for the first time that cytochrome c undergoes a distinct pathway in the alkaline conformational transition from its pro-apoptotic conformational transition, which may have important functional consequences in vivo.

Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide.

Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a alpha-subunit (690 AA) and a heme-binding beta-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5'-triphosphate (GTP) to 3',5'-cyclic guanosine monophosphate (cGMP).

The role of Ile476 in the structural stability and substrate binding of human cytochrome P450 2C8.

The biological function and stability of a cytochrome P450 (CYP) mainly depend on the subtle properties of the residues in the active site cavity, which are generally more divergent among proteins than other parts of the protein. As the most unique member of human CYP2C family, CYP2C8 has an isoleucine (Ile) 476 instead of phenylalanine (Phe) in substrate recognizing site 6 (SRS6). However, the role of Ile476 of CYP2C8 is still unknown.

Tyrosine-67 in cytochrome c is a possible apoptotic trigger controlled by hydrogen bonds via a conformational transition.

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.

Evolutionary alkaline transition in human cytochrome c.

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study.