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.

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein.

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.

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.

In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore.

Phytochrome B (PhyB), one of the major photosensory chromoproteins in plants, mediates a variety of light-responsive developmental processes in a photoreversible manner. To analyze the structural requirements of the chromophore for the spectral properties of PhyB, we have designed and chemically synthesized 20 analogs of the linear tetrapyrrole (bilin) chromophore and reconstituted them with PhyB apoprotein (PHYB). The A-ring acts mainly as the anchor for ligation to PHYB, because the modification of the side chains at the C2 and C3 positions did not significantly influence the formation or difference spectra of adducts. In contrast, the side chains of the B- and C-rings are crucial to position the chromophore properly in the chromophore pocket of PHYB and for photoreversible spectral changes. The side-chain structure of the D-ring is required for the photoreversible spectral change of the adducts. When methyl and ethyl groups at the C17 and C18 positions are replaced with an n-propyl, n-pentyl, or n-octyl group, respectively, the photoreversible spectral change of the adducts depends on the length of the side chains. From these studies, we conclude that each pyrrole ring of the linear tetrapyrrole chromophore plays a different role in chromophore assembly and the photochromic properties of PhyB.

Structure-function relationships of the NMDA receptor antagonist peptide, conantokin-R.

Conantokin-R (con-R) is a gamma-carboxyglutamate-containing 27-residue neuroactive peptide present in the venom of Conus radiatus, and acts as a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor. This peptide features a single disulfide bond, a type of structural element found in most classes of conotoxins, but not in other conantokins. The NMDA receptor antagonist activity of chemically synthesized con-R was determined through an assay involving inhibition of the spermine-enhanced binding of the NMDA receptor channel blocker, [(3)H]MK-801, to rat brain membranes, and yielded an IC(50) of 93 nM. This value represents a 2-5 times better potency than con-G or con-T, the other two characterized conantokins. Circular dichroism (CD) analysis of the metal-free form of con-R is indicative of a low alpha-helical content. There is an increase in alpha-helicity upon the addition of divalent cations, such as Ca(2+), Mg(2+), or Zn(2+). Isothermal titration calorimetry experiments showed one detectable Mg(2+) binding site with a K(d) of 6.5 microM, and two binding sites for Zn(2+), with K(d) values of 150 nM and 170 microM. Residue-specific information of the conformational state of con-R was obtained by two-dimensional (1)H-NMR. Analyses of the alpha-proton chemical shifts, NOE patterns, and hydrogen exchange rates of the peptide indicated an alpha-helical conformation for residues 1-19. Synthetic con-R-derived peptide variants, containing deletions of 7 and 10 amino acid residues from the carboxy-terminus of the wild-type peptide, displayed unaltered cation binding and NMDA receptor antagonist properties. The alpha-helical secondary structures of the two truncation peptides were more stable than full-length con-R, as evidenced by CD measurements and reduced backbone hydrogen exchange rates. These results provide experimental evidence that the structural elements common to the three conantokins thus far identified are the primary determinants for receptor function and cation binding/secondary structure stability.

Total synthesis of horse heart cytochrome c. Preparation and characterization of the (1-66)apofragment.

A peptide corresponding to the native (1-66) sequence of horse heart cytochrome c has been synthesized by stepwise automated solid-phase methods on PAM resin. The course of the synthesis has been monitored by several analytical methods including quantitative ninhydrin and Edman degradation. After HF cleavage, the peptide has been purified by a combination of semipreparative ion-exchange and RP-HPLC. The homogeneity of the purified synthetic peptide has been determined by different criteria including HPLC, amino-acid composition, electrophoresis, antibody binding, tryptic and chymotryptic peptide mapping. After deprotection of the Acm-Cys residues and CNBr cleavage of the Met65-Glu66 peptide bond with simultaneous transformation of the Met65 residue into the activated C-terminal [Hse65]lactone, this purified synthetic peptide has been utilized for conformation-assisted joining experiments in combination with synthetic (66-104) to produce fully synthetic [Hse65]apocytochrome c. This latter, after mitochondria-mediated stereospecific heme insertion, has given a functional molecule corresponding to native horse heart holocytochrome c.

Total synthesis of horse heart cytochrome C.

A strategy based on complexation-assisted condensation of large synthetic protein fragments and mitochondria-mediated stereospecific heme insertion has been utilized to assemble a functional molecule corresponding to native horse heart holocytochrome c. This original approach offers the unique opportunity of selective modifications both in the C-terminal and in the N-terminal regions of the apoprotein and may represent an useful alternative to site-directed mutagenesis, particularly when D-amino acids, chemically and/or isotopically modified or other unnatural amino acids have to be introduced in this important molecule. The present result is an example of how solid phase peptide synthesis of large protein fragments in conjunction with the availability of a specific recognition process may extend the potentiality of the chemical approach to the synthesis of an entire protein.

The use of synthetic peptides in the formation of biophysically and biologically active pulmonary surfactants.

Synthetic pulmonary surfactants consisting of mixtures of phospholipids with synthetic peptides based on the amino acid sequence of human surfactant apoprotein SP-B were prepared. These surfactants were analyzed for their ability to lower surface tension on a pulsating bubble surfactometer and for their capacity to improve lung compliance and increase alveolar expansion in a fetal rabbit model of surfactant deficiency. The data demonstrate that several peptides, ranging from 17 to 45 residues in length, matching the carboxy-terminal sequence of the SP-B protein, when appropriately recombined with the phospholipid dipalmitoylphosphatidylcholine and phosphatidylglycerol (3:1), are capable of producing a synthetic surfactant with biophysical and biologic activity approaching that of human surfactant derived from amniotic fluid.

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.

The solid phase synthesis of a protein activator for lecithin-cholesterol acyltransferase corresponding to human plasma apoC-I.

Apolipoprotein C-I, a protein constituent of the very low density lipoproteins of human plasma, consists of a single chain of 57 amino acids. The total synthesis of a protein corresponding to apolipoprotein C-I in physical properties and compositions was accomplished by solid phase techniques employing a modified polystrene incorporating spacer groups between the point of attachment of the first residue and the polymer matrix. The synthetic apoprotein was shown to activate lecithin:cholesterol acyltransferase to the same extent as the native protein. Comparative lipid-binding studies with dimyristoyl phosphatidylcholine gave complexes for native and synthetic apoprotein which floated at the same density after ultracentrifugation in KBr gradients and had virtually the same lipid:protein ratios.

Chemical synthesis and biochemical properties of peptide fragments of apolipoprotein-alanine.

Apolipoprotein-alanine is an apolipoprotein isolated from very-low-density lipoproteins of human plasma. This protein contains 79 amino acids and binds phosphatidyl choline. Four fragments of this molecule corresponding to sequence positions 41-79 (I), 48-79 (II), 55-79 (III), and 61-79 (IV) have been synthesized by standard solid-phase methods. The resulting peptides were cleaved from the resin and deblocked with liquid HF, then purified by chromatography on Sephadex G-50 and DEAE-cellulose. Each purified peptide eluted as a single, symmetrical peak, exhibited a single band on polyacrylamide gel electrophoresis, and gave an amino-acid analysis in good agreement with the theoretical value. Circular dichroism studies indicated that only fragments I and II became more helical in the presence of phosphatidyl choline and significantly inhibited the reactivation of delipidated beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30), an enzyme that requires phosphatidyl choline for activity. When subjected to ultracentrifugation at density 1.064 g/ml in the presence of phosphatidyl choline, fragments I, II, III, and IV floated to the top of the tube to the extent of 85, 50, 13, and 9%, respectively. These results indicate that residues 55-79 do not contain the minimum determinants required for the binding of phospholipid. However, extension of the peptide's N-terminus by seven residues produces a molecule that does bind phosphatidyl choline.