We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids. The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (Nle) and selenomethionine (SeM). The UV-visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT. All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings. The Se EXAFS of the M121SeM variant is also reported. In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121Nle are 25, 135, and 140 mV, respectively, higher than that of WT azurin. The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center. A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains. Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands.
The electron-transfer reactivity of microperoxidase-8 (MP8), the heme octapeptide derived from enzymatic cleavage of cytochrome c, has been studied by nanosecond flash photolysis methods. Ferric MP8 is rapidly oxidized by photogenerated Ru(bpy)3 3+ in acidic solutions to a ferric cation-radical porphyrin (k ∼ 5.6 × 109 M-1 s-1); the oxidation product in alkaline solutions is ferryl MP8 (k ∼ 2.2 × 109 M-1 s-1). Numerical simulations of the kinetics for the direct oxidation of ferric to ferryl MP8 predict a marked pH effect on the rate of reaction in alkaline solutions; however, only a very weak pH dependence is observed in the range 7−8.5, indicating that the ferryl species is generated by intramolecular electron transfer within a ferric cation-radical porphyrin. Transient spectra taken between pH 6 and 8.5 show increasing ferryl absorption as the pH is increased, demonstrating a pH-dependent equilibrium between the two oxidized forms of MP8.
We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet 7 ]cyt b562, by thioestermediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the ␣-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a fulllength unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet 7 ]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by Ϸ45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments. Because of its unmatched flexibility, total chemical synthesis is emerging as a powerful tool for protein engineering (1, 2). Chemical access to proteins inherently provides the ability to incorporate unnatural amino acids into proteins in a completely general fashion and has opened many new avenues for the study of protein function. The large number and variety of unnatural amino acids available enables researchers to systematically probe size, shape, acidity, hydrogen bonding, and electronic structure effects on the reactivity properties of the protein molecule. Studies of bioinorganic systems in particular can benefit greatly from the ability to place unnatural metal-chelating residues into metalloprotein structures in a site-specific manner.Chemical ligation reactions (3) have been used in the total synthesis of myriad engineered protein targets with unusual backbone and side chain groups. Several ligation chemistries have been used (4-7). In particular, the native chemical ligation (NCL) reaction (8) has enabled the synthesis of many wild-type and engineered protein targets by allowing full-length polypeptide sequences to be built by linking readily obtained smaller unprotected peptide segments with native amide bonds. In the original NCL scheme (9), a thioester-containing peptide reacts in a chemoselective manner with a second peptide that has a cysteine as its N-terminal residue. The side chain thiol of that cysteine undergoes thiol exchange with the thioester moiety of the first peptide. A thioester-linked intermediate is formed, whic...
Microperoxidase-8 (MP8) is the heme octapeptide derived from enzymatic proteolysis of horse-heart cytochrome c. Not only is MP8 a functional peroxidase (it catalyzes the oxidation of various substrates by hydrogen peroxide), it has also served as a useful calibration for the interpretation of the electronic spectroscopic properties of heme proteins. NMR structural characterization of MP8 has been difficult, owing to extensive aggregation at millimolar concentrations. We have obtained well-resolved 1H and 13C NMR spectra of monomeric ferric MP8-CN in mixed aqueous−organic solvent mixtures containing excess cyanide. Most peptide resonances were assigned by through-bond correlations using TOCSY spectra; heme resonances were identified largely by through-space correlations using NOESY spectra. HMQC spectra were interpreted with the aid of proton assignments to identify 13C resonances. Most peptide resonances appear within the diamagnetic region and are very sharp, the exceptions being resonances associated with the His18 residue. Protons on the His18 imidazole ring exhibit very broad resonances, reflecting efficient relaxation. The signals of heme substituents are shifted outside of the diamagnetic envelope, with heme methyl resonances appearing between 10 and 25 ppm. The pattern of MP8-CN heme methyl resonances bears a striking resemblance to those of intact cyanoferric heme c protein derivatives. Large amide-proton/α-proton coupling constants and interresidue NOE contacts were found between residues 14 and 18, while moderate amide-α coupling constants were found between residues 19 and 21. The imidazole group of His18 remains coordinated to the ferric center in MP8; and the pattern of heme methyl resonances confirms that a fixed axial imidazole orientation is preserved in the isolated heme active site. The observed interresidue NOE's and amide−H/Hα proton coupling constants indicate that His18 is part of a rigid loop of five residues anchored to the heme that serves to orient the axial imidazole, while residues 19−21 form a flexible C-terminal domain.
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