Stepwise Evolution Improves Identification of Diverse Peptides Binding to a Protein Target.

Considerable efforts have been made to develop technologies for selection of peptidic molecules that act as substrates or binders to a protein of interest. Here we demonstrate the combination of rational peptide array library design, parallel screening and stepwise evolution, to discover novel peptide hotspots. These hotspots can be systematically evolved to create high-affinity, high-specificity binding peptides to a protein target in a reproducible and digitally controlled process. The method can be applied to synthesize both linear and cyclic peptides, as well as peptides composed of natural and non-natural amino acid analogs, thereby enabling screens in a much diverse chemical space. We apply this method to stepwise evolve peptide binders to streptavidin, a protein studied for over two decades and report novel peptides that mimic key interactions of biotin to streptavidin.

Electronic structure and biologically relevant reactivity of low-spin {FeNO}8 porphyrin model complexes: new insight from a bis-picket fence porphyrin.

Because of HNO's emerging role as an important effector molecule in biology, there is great current interest in the coordination chemistry of HNO and its deprotonated form, the nitroxyl anion (NO(-)), with hemes. Here we report the preparation of four new ferrous heme-nitroxyl model complexes, {FeNO}(8) in the Enemark-Feltham notation, using three electron-poor porphyrin ligands and the bis-picket fence porphyrin H2[3,5-Me-BAFP] (3,5-Me-BAFP(2-) = 3,5-methyl-bis(aryloxy)-fence porphyrin dianion). Electrochemical reduction of [Fe(3,5-Me-BAFP)(NO)] (1-NO) induces a shift of ν(N-O) from 1684 to 1466 cm(-1), indicative of formation of [Fe(3,5-Me-BAFP)(NO)](-) (1-NO(-)), and similar results are obtained with the electron-poor hemes. These results provide the basis to analyze general trends in the properties of ferrous heme-nitroxyl complexes for the first time. In particular, we found a strong correlation between the electronic structures of analogous {FeNO}(7) and {FeNO}(8) complexes, which we analyzed using density functional theory (DFT) calculations. To further study their reactivity, we have developed a new method for the preparation of bulk material of pure heme {FeNO}(8) complexes via corresponding [Fe(porphyrin)](-) species. Reaction of [Fe(To-F2PP)(NO)](-) (To-F2PP(2-) = tetra(ortho-difluorophenyl)porphyrin dianion) prepared this way with acetic acid generates the corresponding {FeNO}(7) complex along with the release of H2. Importantly, this disproportionation can be suppressed when the bis-picket fence porphyrin complex [Fe(3,5-Me-BAFP)(NO)](-) is used, and excitingly, with this system we were able to generate the first ferrous heme-NHO model complex reported to date. The picket fence of the porphyrin renders this HNO complex very stable, with a half-life of ~5 h at room temperature in solution. Finally, with analogous {FeNO}(8) and {FeNHO}(8) complexes in hand, their biologically relevant reactivity toward NO was then explored.

The trans effect of nitroxyl (HNO) in ferrous heme systems: implications for soluble guanylate cyclase activation by HNO.

Soluble guanylate cyclase (sGC) is the primary mammalian nitric oxide (NO) sensor. Through the strong thermodynamic σ-trans effect of NO, binding of NO at the distal side of the ferrous heme induces cleavage of the proximal FeN(His) bond, activating the catalytic domain of the enzyme. It has been proposed that nitroxyl (HNO) is also capable of activating sGC, but the key question remains as to whether HNO can induce cleavage of the FeN(His) bond. Here we report calculated binding constants for 1-methylimidazole (MI) to [Fe(P)(X)] (P=porphine(2-)) where X=NO, HNO, CO, and MI to evaluate the trans interaction of these groups, X, with the proximal imidazole (histidine) in sGC. Systematic assessment of DFT methods suggests that the prediction of accurate MI binding constants is critically dependent on the inclusion of van der Waals interactions (-D functionals). Calculated (B3LYP-D/TZVP) MI binding constants for X=NO and MI are 110 and 5.6 × 10(5)M(-1), respectively, predicted only one order of magnitude higher than the corresponding experimentally determined values. MI binding constants where X=HNO and CO are consistently predicted to be essentially equal and ~six orders of magnitude larger than those of NO, indicating that CO and HNO mediate a weak thermodynamic trans effect in this system. Orbital analysis of the key σ-bonding orbital, π*(h)_d(z2), and comparison of FeN(MI) bond lengths support this prediction. This suggests that HNO does not induce a σ-trans effect strong enough to promote cleavage of the FeN(His) bond-a key step in the activation of sGC.

Ligand recruitment and spin transitions in the solid-state photochemistry of Fe(III)TPPCl.

We report evidence for the formation of long-lived photoproducts following excitation of iron(III) tetraphenylporphyrin chloride (Fe(III)TPPCl) in a 1:1 glass of toluene and CH(2)Cl(2) at 77 K. The formation of these photoproducts is dependent on solvent environment and temperature, appearing only in the presence of toluene. No long-lived product is observed in neat CH(2)Cl(2) solvent. A 2-photon absorption model is proposed to account for the power-dependent photoproduct populations. The products are formed in a mixture of spin states of the central iron(III) metal atom. Metastable six-coordinate high-spin and low-spin complexes and a five-coordinate high-spin complex of iron(III) tetraphenylporphyrin are assigned using structure-sensitive vibrations in the resonance Raman spectrum. These species appear in conjunction with resonantly enhanced toluene solvent vibrations, indicating that the Fe(III) compound formed following photoexcitation recruits a toluene ligand from the surrounding environment. Low-temperature transient absorption (TA) measurements are used to explain the dependence of product formation on excitation frequency in this photochemical model. The six-coordinate photoproduct is initially formed in the high-spin Fe(III) state, but population relaxes into both high-spin and low-spin state at 77 K. This is the first demonstration of coupling between the optical and magnetic properties of an iron-centered porphyrin molecule.

Electronic structure of heme-nitrosyls and its significance for nitric oxide reactivity, sensing, transport, and toxicity in biological systems.

This review summarizes recent developments in the investigation of the electronic structures, spectroscopic properties, and reactivities of ferrous and ferric heme-nitrosyls and how this relates to important biological processes. Ferrous heme-nitrosyls show interesting variations in electronic structure as a function of the different types of proximal ligands, as is evident from electron paramagnetic resonance, magnetic circular dichroism, and vibrational spectroscopy. In particular, coordination of imidazoles like histidine (His) increases the radical character on NO and, in this way, could help activate the bound NO for catalysis. Vice versa, the bound NO ligand imposes a strong sigma trans effect on the proximal His, which, in the case of soluble guanylate cyclase (sGC), the biological NO sensor protein, induces breaking of the Fe(II)-His bond and activates the protein. The possibility of sGC activation by HNO is also discussed. Finally, the properties of ferrous heme-nitrosyls with proximal cysteinate (Cys) coordination are evaluated. It has been known for some time that ferric heme-nitrosyls are intrinsically more labile than their ferrous counterparts, but the underlying reasons for this observation have not been clarified. New results show that this property relates to the presence of a low-lying excited state that is dissociative with respect to the Fe(III)-NO bond. On the other hand, the ground state of these complexes is best described as Fe(II)-NO(+), which shows a very strong Fe-NO bond, as is evident from vibrational spectroscopy. NO, therefore, is a weak ligand to ferric heme, which, at the same time, forms a strong Fe-NO bond. This is possible because the thermodynamic weakness and spectroscopic strength of the Fe-NO bond relate to the properties of different electronic states. Thiolate coordination to ferric hemes leads to a weakening of both the Fe-NO and N-O bonds as a function of the thiolate donor strength. This observation can be explained by a sigma backbond into the sigma* orbital of the Fe-N-O unit that is mediated by the thiolate sigma-donor orbital via orbital mixing. This is a new interaction in heme-nitrosyl that has not been observed before. This also induces a bending of the Fe-N-O subunit in these cases. New spectroscopic data on a corresponding model complex are included in this paper. Finally, the mechanism of NO reduction by cytochrome P450nor is elucidated based on recent density functional theory results.

Five- and six-coordinate adducts of nitrosamines with ferric porphyrins: structural models for the Type II interactions of nitrosamines with ferric cytochrome P450.

Nitrosamines are well-known for their toxic and carcinogenic properties. The metabolic activation of nitrosamines occurs via interaction with the heme-containing cytochrome P450 enzymes. We report the preparation and structural characterization of a number of nitrosamine adducts of synthetic iron porphyrins. The reactions of the cations [(por)Fe(THF)(2)]ClO(4) (por = TPP, TTP, OEP) with dialkylnitrosamines (R(2)NNO; R(2) = Me(2), Et(2), (cyclo-CH(2))(4), (cyclo-CH(2))(5), (PhCH(2))(2)) in toluene generate the six-coordinate high-spin (S = 5/2) [(por)Fe(ONNR(2))(2)]ClO(4) compounds and a five-coordinate intermediate-spin (S = 3/2) [(OEP)Fe(ONNMe(2))]ClO(4) derivative in 57-72% yields (TPP = 5,10,15,20-tetraphenylporphyrinato dianion, TTP = 5,10,15,20-tetra-p-tolylporphyrinato dianion, OEP = 2,3,7,8,12,13,17,18-octaethylporphyrinato dianion). The N-O and N-N vibrations of the coordinated nitrosamine groups in [(por)Fe(ONNR(2))(2)]ClO(4) occur in the 1239-1271 cm(-1) range. Three of the six-coordinate [(por)Fe(ONNR(2))(2)]ClO(4) compounds and one five-coordinate [(OEP)Fe(ONNMe(2))]ClO(4) compound have been characterized by single crystal X-ray crystallography. All the nitrosamine ligands in these complexes bind to the ferric centers via a sole eta(1)-O binding mode. No arylnitrosamine adducts were obtained from the reactions of the precursor compounds [(por)Fe(THF)(2)]ClO(4) with three arylnitrosamines (Ph(2)NNO, Ph(Me)NNO, Ph(Et)NNO). However, prolonged exposure of [(por)Fe(THF)(2)]ClO(4) to these arylnitrosamines resulted in the formation of the known five-coordinate (por)Fe(NO) derivatives. The latter (por)Fe(NO) compounds were obtained more readily by the reactions of the three arylnitrosamines with the four-coordinate (por)Fe(II) precursors.

Iron-porphyrin NO complexes with covalently attached N-donor ligands: formation of a stable six-coordinate species in solution.

A series of substituted tetraphenylporphyrin type macrocycles (TMP or To-F(2)PP) with covalently attached N-donor ligands (pyridine or imidazole linker) have been synthesized. Linkers with varying chain lengths and designs have been applied to systematically investigate the effect of chain length and rigidity on the binding affinity of the linker to the corresponding Fe(II)-NO heme complexes. The binding of the linker is monitored in solution using a variety of spectroscopic methods including UV-vis absorption, EPR, and IR spectroscopy. Both the N-O stretching frequency and the imidazole (14)N hyperfine coupling constants show a good correlation with the Fe-(N-donor) bond strength in these systems. The complexes with covalently attached pyridyl and alkyl imidazole ligands only exhibit weak interactions of the linker with iron(II). However, the stable six-coordinate complex [Fe(To-F(2)PP-BzIM)(NO)] (4) is obtained when a rigid benzyl linker is applied. This complex exhibits typical properties of six-coordinate ferrous heme-nitrosyls in which an N-donor ligand is bound trans to NO, including the Soret band at 427 nm and the typical nine line (14)N hyperfine splitting in the EPR spectrum. A crystal structure has been obtained for the corresponding zinc complex. Here, we report the first systematic study on the requirements for the formation of stable six-coordinate ferrous heme nitrosyl complexes in solution at room temperature in the absence of excess axial N-donor ligand.

Vibrational assignments of six-coordinate ferrous heme nitrosyls: new insight from nuclear resonance vibrational spectroscopy.

This Communication addresses a long-standing problem: the exact vibrational assignments of the low-energy modes of the Fe-N-O subunit in six-coordinate ferrous heme nitrosyl model complexes. This problem is addressed using nuclear resonance vibrational spectroscopy (NRVS) coupled to (15)N(18)O isotope labeling and detailed simulations of the obtained data. Two isotope-sensitive features are identified at 437 and 563 cm(-1). Normal coordinate analysis shows that the 437 cm(-1) mode corresponds to the Fe-NO stretch, whereas the 563 cm(-1) band is identified with the Fe-N-O bend. The relative NRVS intensities of these features determine the degree of vibrational mixing between the stretch and the bend. The implications of these results are discussed with respect to the trans effect of imidazole on the bound NO. In addition, a comparison to myoglobin-NO (Mb-NO) is made to determine the effect of the Mb active site pocket on the bound NO.

Dynamics of dilute water in carbon tetrachloride.

A dilute solution of water in a hydrophobic solvent, such as carbon tetrachloride (CCl4), presents an opportunity to study the rotational properties of water without the complicating effects of hydrogen bonds. We report here the results of theoretical, experimental, and semiempirical studies of a 0.03 mole percent solution of water in CCl4. It is shown that for this solution there are negligible water-water interactions or water-CCl4 interactions; theoretical and experimental values for proton NMR chemical shifts (deltaH) are used to confirm the minimal interactions between water and the CCl4. Calculated ab initio values and semiempirical values for oxygen-17 and deuterium quadrupole coupling constants (chi) of water/CCl4 clusters are reported. Experimental values for the 17O, 2H, and 1H NMR spin-lattice relaxation times, T1, of 0.03 mole percent water in dilute CCl4 solution at 291 K are 94+/-3 ms, 7.0+/-0.2 s, and 12.6+/-0.4 s, respectively. These T1 values for bulk water are also referenced. "Experimental" values for the quadrupole coupling constants and relaxation times are used to obtain accurate, experimental values for the rotational correlation times for two orthogonal vectors in the water molecule. The average correlation time, tauc, for the position vector of 17O (orthogonal to the plane of the molecule) in monomer water, H2(17)O, is 91 fs. The average value for the deuterium correlation time for the deuterium vector in 2H2O is 104 fs; this vector is along the OD bond. These values indicate that the motion of monomer water in CCl4 is anisotropic. At 291 K, the oxygen rotational correlation time in bulk 2H2(17)O is 2.4 ps, the deuterium rotational correlation time in the same molecule is 3.25 ps. (Ropp, J.; Lawrence, C.; Farrar, T. C.; Skinner, J. L. J. Am. Chem. Soc. 2001, 123, 8047.) These values are a factor of about 20 longer than the tauc value for dilute monomer water in CCl4.