De novo design of an artificial bis[4Fe-4S] binding protein.

In nature, protein subunits containing multiple iron-sulfur clusters often mediate the delivery of reducing equivalents from metabolic pathways to the active site of redox proteins. The de novo design of redox active proteins should include the engineering of a conduit for the delivery of electrons to and from the active site, in which multiple redox active centers are arranged in a controlled manner. Here, we describe a designed three-helix protein, DSD-bis[4Fe-4S], that coordinates two iron-sulfur clusters within its hydrophobic core. The design exploits the pseudo two-fold symmetry of the protein scaffold, DSD, which is a homodimeric three-helix bundle. Starting from the sequence of the parent peptide, we mutated eight leucine residues per dimer in the hydrophobic core to cysteine to provide the first coordination sphere for cubane-type iron-sulfur clusters. Incorporation of two clusters per dimer is readily achieved by in situ reconstitution and imparts increased stability to thermal denaturation compared to that of the apo form of the peptide as assessed by circular dichroism-monitored thermal denaturation. The presence of [4Fe-4S] clusters in intact proteins is confirmed by UV-vis spectroscopy, gel filtration, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy. Pulsed electron-electron double-resonance experiments have detected a magnetic dipole interaction between the two clusters ~0.7 MHz, which is consistent with the expected intercluster distance of 29-34 Å. Taken together, our data demonstrate the successful design of an artificial multi-iron-sulfur cluster protein with evidence of cluster-cluster interaction. The design principles implemented here can be extended to the design of multicluster molecular wires.

Rational design of a supra C-1027: kinetically stabilized analogue of the antitumor enediyne chromoprotein.

C-1027, an extremely potent antitumor agent, is composed of a highly reactive chromophore and an apoprotein. While the chromophore causes DNA cleavage, the apoprotein functions as its carrier. Despite these ideal properties as an anticancer agent, C-1027 slowly self-decomposes through chromophore-mediated abstraction of hydrogens from the apoprotein. In this paper, we report the design and preparation of an engineered C-1027 apoprotein that decelerates this self-decomposition pathway. Our design is based on the kinetic isotope effect, and deuterium is incorporated instead of protium into the hydrogen-abstraction site. The deuterated supra C-1027 was found to have a 4-fold longer lifetime than the natural C-1027.

Development of a pharmaceutical apotransferrin product for iron binding therapy.

High-dose chemotherapy of patients with haematological malignancies results in extracellular iron accumulation and appearance of non-transferrin-bound iron, which is thought to predispose the patients to septic infections and contribute to organ toxicity. We describe the development of a human plasma-derived apotransferrin product for iron binding therapy. The product is purified from Cohn fraction IV of human plasma by two ion exchange chromatography steps and ultrafiltration. The process comprises solvent detergent treatment as the main virus inactivation step and 15 nm virus filtration and polyethylene glycol precipitation as removal steps for physico-chemically resistant infectious agents. Product characterization by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. The purity of the product was more than 98%, main impurities being IgG, IgA and hemopexin. The product had intact iron binding capacity and native conformation. A stable liquid formulation for the finished product was developed. The product has proved safe and well tolerated in early clinical trials in iron binding therapy.

A bacterial c-type cytochrome can be translocated to the periplasm as an apo form; the biosynthesis of cytochrome cd1 (nitrite reductase) from Paracoccus denitrificans.

An apo form of cytochrome cd1 (nitrite reductase) of Paracoccus denitrificans has been detected immunologically in the periplasm of a mutant that lacks all c-type cytochromes. A method for the preparation of apo-nitrite reductase (lacking both c- and d-type haem) from the holoenzyme of wild-type cells has been developed. The apoprotein synthesized by the mutant is indistinguishable from the chemically prepared apoprotein in respect of: (i) subunit molecular weight; (ii) formation of a homodimer; (iii) properties on anion exchange chromatography. The holoenzyme has similar properties in respect of (i) and (ii) but behaves differently during anion exchange. A suggested mode of assembly of cytochrome cd1 is translocation into the periplasm of a precursor polypeptide, maturation by a signal peptidase to give an apoprotein identical to that prepared chemically from the holoenzyme, followed by insertion of c-type and d-type haem in an as yet unknown order.