Unnatural amino acids in the synthesis and semisynthesis of metalloprotein motifs

The design, synthesis, and characterization of two novel metalloprotein motifs is presented. The first project involved the design and construction of a protein motif which was programmed to form a tetradentate metal complex upon the addition of metal cations. The overall structure of the motif was based on a ββ super-secondary structure consisting of a flexible peptide sequence flanked by metal binding regions located at the carboxy and amino termini. The metal binding region near the amino terminus was constructed from a reverse turn motif with two metal ligating residues, (2R, 3R)-β-methyl-cysteine and histidine. Selection of the peptide sequence for this region was based on the conformational analysis of a series of tetrapeptides designed to form reverse turns in solution.

The stereospecific syntheses of a series of novel bipyridyl- and phenanthrolylsubstituted amino acids was carried out to provide ligands for the carboxy terminus metal binding region. These residues were incorporated into peptide sequences using solid phase peptide synthesis protocols, and metal binding studies indicated that the metal binding properties of these ligands was dictated by the specific regioisomer of the heteroaromatic ring and the peptide primary sequence.

Finally, a peptide containing optimized components for the metal binding regions was prepared to test the ability of the compound to form the desired intramolecular peptide:metal cation complexes. Metal binding studies demonstrated that the peptide formed monomeric complexes with very high metal cation binding affinities and that the two metal binding regions act cooperatively in the metal binding process. The use of these systems in the design of proteins capable of regulating naturally occurring proteins is discussed.

The second project involved the semisynthesis of two horse heart cytochrome c mutants incorporating the bipyridyl-amino acids at position 72 of the protein sequence. Structural studies on the proteins indicated that the bipyridyl amino acids had a neglible effect on the protein structure. One of the mutants was modified with Ru(bpy)_2^(+2) to form a redox-active protein, and the modified protein was found to have enhanced electron transfer properties between the heme and the introduced metal site.

Structurally engineered cytochromes c with novel ligand-binding properties

Semisynthesis of horse heart cytochrome c and site-directed mutagenesis of Saccharomyces cerevisiae (S. c.) iso-1-cytochrome c have been utilized to substitute Ala for the cytochrome c heme axial ligand Met80 to yield ligand-binding proteins (horse heart Ala80cyt c and S.c. Ala80cyt c) with spectroscopic properties remarkably similar to those of myoglobin. Both species of Fe(II)Ala80cyt c form exceptionally stable dioxygen complexes with autoxidation rates 10-30x smaller and O₂ binding constants ~ 3x greater than those of myoglobin. The resistance of O₂-Fe(II)Ala80cyt c to autoxidation is attributed in part to protection of the heme site from solvent as exhibited by the exceptionally slow rate of CO binding to the heme as well as the low quantum yield of CO photodissociation.

UV/vis, EPR, and paramagnetic NMR spectroscopy indicate that at pH 7 the Fe(III)Ala80cyt c heme is low-spin with axial His-OH^- coordination and that below pH ~6.5, Fe(III)Ala80cyt cis high-spin with His-H₂O heme ligation. Significant differences in the pH dependence of the ¹H NMR spectra of S.c. Fe(III)Ala80cyt c compared to wild-type demonstrate that the axial ligands influence the conformational energetics of cytochrome c.

¹H NMR spectroscopy has been utilized to determine the solution structure of the cyanide derivative of S.c. Fe(III)Ala80cyt c. 82% of the resonances in the ¹H NMR spectrum of S.c. CN-Fe(III)Ala80cyt c have been assigned through 1D and 2D experiments. The RMSD values after restrained energy minimization of the family of 17 structures obtained from distance geometry calculations are 0.68 ± 0.11 Å for the backbone and 1.32 ± 0.14 Å for all heavy atoms. The solution structure indicates that a tyrosine in the "distal" pocket of CN-Fe(III)Ala80cyt c forms a hydrogen bond with the Fe(III)-CN unit, suggesting that it may play a role analogous to that of the distal histidine in myoglobin in stabilizing the dioxygen adduct.