No abstract

Small proteins or protein domains generally require disulfide bridges or metal sites for their stabilization. Here it is shown that the beta beta alpha architecture of zinc fingers can be reproduced in a 23-residue polypeptide in the absence of metal ions. The sequence was obtained through an iterative design process. A key feature of the final design is the incorporation of a type II' beta turn to aid in beta-hairpin formation. Nuclear magnetic resonance analysis reveals that the alpha helix and beta hairpin are held together by a defined hydrophobic core. The availability of this structural template has implications for the development of functional polypeptides.

We examined the hydration of amides of ␣ 3 D, a simple, designed three-helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy (∼ 50%-70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide IЈ band) of a group of surface Ala residues. The surface-exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide IЈ bands of the buried 13 C-labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen-deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4-7 residues Nterminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 3 10 /␣/-helical conformation.

Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. To gain further insight into the effect of these highly fluorinated amino acids on helix formation exclusively, we measured the helix propensity of three highly fluorinated amino acids: (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), and (S)-pentafluorophenylalanine (Pff). We have developed a short chemoenzymatic synthesis of Hfl with extremely high enantioselectivity (>99%). To measure the helix propensity (w) of the amino acids, alanine-based peptides were synthesized, purified, and investigated by circular dichroism spectroscopy (CD). On the basis of the CD data, the helix propensity of hydrocarbon amino acids can decrease up to 24-fold (1.72 kcal.mol-1.residue-1) upon fluorination. This difference in helix propensity has previously been overlooked in estimating the magnitude of the fluoro-stabilization effect (which has been estimated to be 0.32-0.83 kcal.mol-1.residue-1 for Hfl), resulting in a gross underestimation. Therefore, the full potential of the fluoro-stabilization effect should provide even more stable proteins than the fluoro-stabilized proteins to date.

Bonding interactions between the iron and the porphyrin macrocycle of five- and six-coordinate high-spin iron(III)-porphyrin complexes are analyzed within the framework of approximate density functional theory with the use of the quantitative energy decomposition scheme in combination with removal of the vacant pi orbitals of the porphyrin from the valence space. Although the relative extent of the iron-porphyrin interactions can be evaluated qualitatively through the spin population and orbital contribution analyses, the bond strengths corresponding to different symmetry representations can be only approximated quantitatively by the orbital interaction energies. In contrast to previous suggestions, there are only limited Fe --> P pi back-bonding interactions in high-spin iron(III)-porphyrin complexes. It is the symmetry-allowed bonding interaction between d(z)2 and a(2u) orbitals that is responsible for the positive pi spin densities at the meso-carbons of five-coordinate iron(III)-porphyrin complexes. Both five- and six-coordinate complexes show significant P --> Fe pi donation, which is further enhanced by the movement of the metal toward the in-plane position for six-coordinate complexes. These bonding characteristics correlate very well with the NMR data reported experimentally. The extraordinary bonding interaction between d(z)2 and a(2u) orbitals in five-coordinate iron(III)-porphyrin complexes offers a novel symmetry-controlled mechanism for spin transfer between the axial ligand sigma system and the porphyrin pi system and may be critical to the electron transfer pathways mediated by hemoproteins.

(2,3,7,8,12,13,17,18-Octaethyl-5,10,15,20-tetraphenylporphinato)iron(III) chloride FeIII(OETPP)Cl and (2,3,7,8,12,13,17,18-octamethyl-5,10,15,20-tetraphenylporphinato)iron(III) chloride FeIII(OMTPP)Cl complexes have been synthesized and characterized by 1H NMR and X-ray crystallography. Both molecules are severely nonplanar and assume saddle shapes in solid state. Variable-temperature 1H NMR studies confirm that the conformational distortions are maintained in solution with ΔG ⧧ = 15.8 and 10.1 kcal/mol for ring inversion for Fe(OETPP)Cl and Fe(OMTPP)Cl, respectively. EPR (g ⊥ = 5.2−5.3 at 77 K), magnetic moments (μeff = 4.7−5.2 μB at 300 K), and structural data (Fe−Np = 2.03 Å, Fe−Cl = 2.24−2.25 Å) all indicate that unlike high-spin FeIII(TPP)Cl and FeIII(OEP)Cl (S = 5/2), FeIII(OETPP)Cl and FeIII(OMTPP)Cl these complexes are of the uncommon quantum-mixed S = 5/2, 3/2 intermediate-spin state. Saddle-shaped ring deformations lower the symmetries of the complexes into C 2v . Other than the nonaxial symmetric EPR spectra of both complexes, 1H NMR spectrum of FeIII(OETPP)Cl shows large asymmetry to the methylene proton shifts. Certain cytochromes c‘ from photosynthetic bacteria reported to be of similar quantum-mixed intermediate-spin and showed EPR signals of rhombic symmetry have been noted to be with saddle-shaped deformations. These anomalous spin states and electronic structure asymmetry are ascribed to the ring deformation of the porphyrin macrocycle.

No abstract

The ability to tune the metal binding affinity of small peptides through the incorporation of unnatural multidentate α-amino acids and the preorganization of peptide structure is illustrated. Herein, we describe the exploitation of a family of α-amino acids that incorporate powerful bidentate ligands (bipyridyl and phenanthrolyl groups) as integral constituents of the residues' side chains. The residues involved are the 6-, 5-, and 4-substituted (S)-2-amino-3-(2,2‘-bipyridyl)propanoic acids (1, 6Bpa; 2, 5Bpa; 3, 4Bpa), (S)-2-amino-3-(1,10-phenanthrol-2-yl)propanoic acid (4, Fen), and a novel neocuproine-containing α-amino acid, (S)-2-amino-3-(9-methyl-1,10-phenanthrol-2-yl)propanoic acid (5, Neo). Within this family of amino acids, variations in metal binding due to the nature of the ring system (2,2‘-bipyridyl or 1,10-phenanthrolyl) and the point of attachment to the amino acid β-carbon are observed. Additionally, the underlying peptide architecture significantly influences binding for peptides that include multiple metal-ligating residues. These differences in affinity arise from the interplay of ligand type and structural preorganization afforded by the peptide sequence, resulting in dissociation constants ranging from 10-3 to <10-6 M for ZnII. These studies illustrate that significant control of metal cation binding affinity, preference, and stoichiometry may be achieved through the use of a wide variety of native and unnatural metal-coordinating amino acids incorporated into a polypeptide architecture.