Copper-mediated peptide arylation selective for the N-terminus

Polypeptides present remarkable selectivity challenges for chemical methods. Amino groups are ubiquitous in polypeptide structure, yet few paradigms exist for reactivity and selectivity in arylation of amine groups. This communication describes the utilization of boronic acid reagents bearing certain o-electron withdrawing groups for copper-mediated amine arylation of the N-terminus under mild conditions and primarily aqueous solvent. The method adds to the toolkit of boronic acid reagents for polypeptide modification under mild conditions in water that shows complete selectivity for the N-terminus in the presence of lysine side chains.

The discovery of unique Chan-Lam coupling reactivity of arylboronic acids containing an ortho-sulfonamide group allows site-specific tailoring of peptide structure.     

The metal selectivity of a short peptide maquette imitating the high-affinity metal-binding site of E. coli HypB

A seven-residue peptide based on the high-affinity metal-binding site of E. coli HypB maintains the nickel-binding activity of the full-length protein. The ability of the peptide to bind transition metals other than nickel was explored, and is discussed in the context of the function of HypB in hydrogenase biosynthesis.     

An N-protection free ligation of the peptide thioester and the peptide with an N-alkoxy- or N-aryloxyamino group at its N-terminus

Peptides with an N-alkoxy or N-aryloxy amino acid at their N-terminus were synthesized and successfully ligated with a peptide thioester by silver ion activation under a slightly acidic condition without requiring protection of the side chain amino groups. The N-methoxy group was easily cleaved by the SmI₂ reduction in CH₃OH aq. to obtain the desired peptide with a native peptide bond. This method was successfully applied to the synthesis of the human atrial natriuretic peptide showing the efficiency of the novel ligation.     

A perturbed pK(a) at the binding site of the nicotinic acetylcholine receptor: implications for nicotine binding

A series of tethered quaternary ammonium derivatives of Tyr have been incorporated into the binding site of the nicotinic acetylcholine receptor (nAChR) using the in vivo nonsense suppression method, producing constitutively active (self-gating) receptors. We have incorporated primary, secondary, and tertiary amine tethered agonists to give receptors whose constitutive activity can be modulated by pH. Lowering the pH protonates the tethered amine, giving it a positive charge and allowing it to reversibly activate the receptor. Tertiary and secondary tethered amines, TyrO3T and TyrO3S, have been successfully incorporated at alpha149 in the nAChR. Constitutive currents at pH 5.5 are 6 times those at pH 9.0. The pKa of TyrO3T in the binding site appears to be 6 or lower, differing substantially from its pKa in solution ( approximately 9.3). This local pKa perturbation has substantial implications for pharmacological research on the nAChR: of the tertiary agonists considered, noracetylcholine experiences this pKa perturbation, while nicotine does not.     

A peptide dendrimer enzyme model with a single catalytic site at the core

Catalytic esterase peptide dendrimers with a core active site were discovered by functional screening of a 65,536-member combinatorial library of third-generation peptide dendrimers using fluorogenic 1-acyloxypyrene-3,6,8-trisulfonates as substrates. In the best catalyst, RMG3, ((AcTyrThr)(8)(DapTrpGly)(4)(DapArgSerGly)(2)DapHisSerNH2), ester hydrolysis is catalyzed by a single catalytic histidine residue at the dendrimer core. A pair of arginine residues in the first-generation branch assists substrate binding. The catalytic proficiency of dendrimer RMG3 (kcat/KM = 860 M(-1) min(-1) at pH 6.9) per catalytic site is comparable to that of the multivalent esterase dendrimer A3 ((AcHisSer)(8)(DapHisSer)(4)(DapHisSer)2DapHisSerNH2) which has fifteen histidines and five catalytic sites (Delort, E. et al. J. Am. Chem. Soc. 2004, 126, 15642-15643). Remarkably, catalysis in the single site dendrimer RMG3 is enhanced by the outer dendritic branches consisting of aromatic amino acids. These interactions take place in a relatively compact conformation similar to a molten globule protein as demonstrated by diffusion NMR. In another dendrimer, HG3 ((AcIlePro)(8)(DapIleThr)(4)(DapHisAla)(2)DapHisLeuNH2) by contrast, catalysis by a core of three histidine residues is unaffected by the outer dendritic layers. Dendrimer HG3 or its core HG1 exhibit comparable activity to the first-generation dendrimer A1 ((AcHisSer)(2)DapHisSerNH2). The compactness of dendrimer HG3 in solution is close to that a denatured peptide. These experiments document the first esterase peptide dendrimer enzyme models with a single catalytic site and suggest a possible relationship between packing and catalysis in these systems.     

Evidence for oxygen binding at the active site of particulate methane monooxygenase

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an α(3)β(3)γ(3) trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit (denoted as spmoB) indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O(2) or H(2)O(2) or pMMO with H(2)O(2), an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the μ-η(2):η(2)-peroxo-Cu(II)(2) species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O(2) binding to the proposed active site. Reaction of the 345 nm species with CH(4) results in the disappearance of the spectroscopic feature, suggesting that this O(2) intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site.     

Thermodynamic analysis of a hydrophobic binding site: probing the PDZ domain with nonproteinogenic peptide ligands

[structure: see text] Isothermal titration calorimetry (ITC) is used to study the thermodynamic consequences of systematically modifying the hydrophobic character of a single residue in a series of protein-binding ligands. By substituting standard and nonproteinogenic aliphatic amino acids for the C-terminal valine of the hexapeptide KKETEV, binding to the third PDZ domain (PDZ3) of the PSD-95 protein is characterized by distinct changes in the Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) parameters. One notable observation is that peptide binding affinity can be improved with a nonstandard residue.     

基于大环多胺的新型线型聚合物的合成及与DNA相互作用的研究

设计合成了一种新型线形大环聚合多胺（POGEC）,该聚合物可通过1,3-丙二醇二缩水甘油基醚和1,7-二（二乙氧磷酰基）-1,4,7,10-四氮杂环十二烷反应得到．琼脂糖凝胶电泳和电子扫描光谱（SEM）显示POGEC与DNA有很强的结合能力．当生理浓度的氯化钠溶液加入到已经形成的POGEC／DNA复合物中,DNA可被有效释放．通过荧光光谱测量了POGEC与DNA的结合能力及紧缩能力．圆二色光谱表明POGEC的强结合力并未改变DNA的构象．     

Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction between 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane. High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA condensation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC. Supported by the National Natural Science Foundation of China (Grant Nos. 20725206 and 20732004), Specialized Research Fund for the Doctoral Program of Higher Education, and Scientific Fund of Sichuan Province for Outstanding Young Scientist     

Probing the copper(II) binding features of angiogenin. Similarities and differences between a N-terminus peptide fragment and the recombinant human protein

The angiogenin protein (hAng) is a potent angiogenic factor and its cellular activities may be affected by copper ions even if it is yet unknown how this metal ion is able to produce this effect. Among the different regions of hAng potentially able to bind copper ions, the N-terminal domain appears to be an ideal candidate. Copper(II) complexes of the peptide fragments encompassing the amino acid residues 4-17 of hAng protein were characterized by potentiometric, UV-vis, CD, and EPR spectroscopic methods. The results show that these fragments have an unusual copper(II) binding ability. At physiological pH, the prevailing complex species formed by the peptide encompassing the protein sequence 4-17 is [CuHL], in which the metal ion is bound to two imidazole and two deprotonated amide nitrogen atoms disposed in a planar equatorial arrangement. Preliminary spectroscopic (UV-vis, CD, and EPR) data obtained on the copper(II) complexes formed by the whole recombinant hAng protein, show a great similarity with those obtained for the N-terminal peptide fragments. These findings indicate that within the N-terminal domain there is one of the preferred copper(II) ions anchoring site of the whole recombinant hAng protein.     

Geometrically feasible binding modes of a flexible ligand molecule at the receptor site

A very efficient algorithm for determining the geometrically feasible binding modes of a flexible ligand molecule at the receptor site is presented. It is based on distance geometry but maintains the requirements of three dimensions. The distance geometry manipulation can superimpose two bodies without explicitly calculating the necessary rigid rotation and translation. The whole conformation space of a flexible molecule can be efficiently examined by considering only a finite number of conformational points. The method is suitable only when the criterion for superposition is some minimum distance limit. It cannot, however, give the exact distance between two points in two different bodies.     

Self-assembled multivalency: dynamic ligand arrays for high-affinity binding

Multivalency is a powerful strategy for achieving high-affinity molecular recognition in biological systems. Recently, attention has begun to focus on using self-assembly rather than covalent scaffold synthesis to organize multiple ligands. This approach has a number of advantages, including ease of synthesis/assembly, tunability of nanostructure morphology and ligands, potential to incorporate multiple active units, and the responsive nature of self-assembly. We suggest that self-assembled multivalency is a strategy of fundamental importance in the design of synthetic nanosystems to intervene in biological pathways and has potential applications in nanomedicine.     

Peptides that mimic glycosaminoglycans: high-affinity ligands for a hyaluronan binding domain.

BACKGROUND: Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) that promotes motility, adhesion, and proliferation in mammalian cells, as mediated by cell-surface HA receptors. We sought to identify non-carbohydrate ligands that would bind to and activate cell-surface HA receptors. Such analogs could have important therapeutic uses in the treatment of cancer, wound healing, and arthritis, since such ligands would be resistant to degradation by hyaluronidase (HAse). RESULTS: Peptide ligands that bind specifically to the recombinant HA binding domain (BD) of the receptor for hyaluronan-mediated motility (RHAMM) were obtained by screening two peptide libraries: (i) random 8-mers and (ii) biased 8-mers with alternating acidic side chains, i.e. XZXZXZXZ (X=all-L-amino acids except Cys, Lys, or Arg; Z=D-Asp, L-Asp, D-Glu, or L-Glu). Selectivity of the peptide ligands for the HABD was established by (i) detection of binding of biotin- or fluorescein-labeled peptides to immobilized proteins and (ii) fluorescence polarization of FITC-labeled peptides with the HABD in solution. HA competitively displaced binding of peptides to the HABD, while other GAGs were less effective competitors. The stereochemistry of four biased octapeptides was established by synthesis of the 16 stereoisomers of each peptide. Binding assays demonstrated a strong preference for alternating D and L configurations for the acidic residues, consistent with the calculated orientation of glucuronic acid moieties of HA. CONCLUSIONS: Two classes of HAse-resistant peptide mimetics of HA were identified with high affinity, HA-compatible binding to the RHAMM HABD. This demonstrated that non-HA ligands specific to a given HA binding protein could be engineered, permitting receptor-specific targeting.     

Electrochemical quantification of high-affinity halide binding by a steroid-based receptor

The strength of binding between a cholapod receptor and halide anions is quantified using voltammetry at the liquid/liquid interface, revealing very high affinities and size-selectivity peaking at chloride.     

Histidine-rich peptide: evidence for a single zinc-binding site on H5WYG peptide that promotes membrane fusion at neutral pH

The histidine-rich peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) was found to induce membrane fusion at physiologic pH in the presence of zinc chloride. In this study, we examined the ion selectivity of the interaction of Zn(2+) with H5WYG. This investigation was conducted by using adsorption at air/water interface and mass spectrometry. We found that a peptide-metal complex is formed with Zn(2+) ions. Electrospray ionisation-mass spectrometry (ESI-MS) reveals that the [H5WYG + Zn + 2H](4+), [H5WYG + Zn + H](3+) and [H5WYG + Zn](2+) ions, appearing by increasing the amount of Zn(2+) equivalent, correspond to a monomolecular H5WYG - Zn(2+) complex. Tandem mass spectrometry (MS/MS) provides evidence for the binding of the single Zn(2+) ion to the H(11) and H(19) and probably H(15) residues.     

Copper(II) complex formation with a linear peptide encompassing the putative cell binding site of angiogenin

Angiogenin is one of the more potent angiogenic factors known, whose activity may be affected by the presence of copper ions. Copper(II) complexes with the peptides encompassing the putative endothelial cell binding domain of angiogenin, Ac-KNGNPHREN-NH(2) and Ac-PHREN-NH(2), have been characterized by potentiometric, UV-vis, CD and EPR spectroscopic methods. The coordination features of all the copper complex species derived by both peptides are practically the same, as predictable because of the presence of a proline residue within their aminoacidic sequence. In particular, Ac-PHREN-NH(2) is really the aminoacidic sequence involved in the binding to copper(II). Thermodynamic and spectroscopic evidence are given that side chain oxygen donor atom of glutamyl residue is involved in the copper binding up to physiological pH. EPR parameters suggest that the carboxylate group is still involved also in the predominant species [Cu(L)H(-2)], the metal coordination environment being probably formed by N(Im), 2N(-), H(2)O in equatorial plane and an oxygen atom from COO(-) in apical position, or vice versa, with the carboxylate oxygen atom in the copper coordination plane and the water molecule confined to one of the apical positions. Moreover, the comparison with the thermodynamic and spectroscopic results in the case of the copper(ii) complex species formed by the single point mutated peptide, Ac-PHRQN-NH(2), provides further evidence of the presence of carboxylate oxygen atom in the copper coordination sphere.     

Biomimetic nanostructures: creating a high-affinity zinc-binding site in a folded nonbiological polymer

One of the long-term goals in developing advanced biomaterials is to generate protein-like nanostructures and functions from a completely nonnatural polymer. Toward that end, we introduced a high-affinity zinc-binding function into a peptoid (N-substituted glycine polymer) two-helix bundle. Borrowing from well-understood zinc-binding motifs in proteins, thiol and imidazole moieties were positioned within the peptoid such that both helices must align in close proximity to form a binding site. We used fluorescence resonance energy transfer (FRET) reporter groups to measure the change of the distance between the two helical segments and to probe the binding of zinc. We systematically varied the position and number of zinc-binding residues, as well as the sequence and size of the loop that connects the two helical segments. We found that certain peptoid two-helix bundles bind zinc with nanomolar affinities and high selectivity compared to other divalent metal ions. Our work is a significant step toward generating biomimetic nanostructures with enzyme-like functions.     

Pyrene.pyrene complexes at the active site of cytochrome P450 3A4: evidence for a multiple substrate binding site

Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates.     

The alpha-helix folds more rapidly at the C-terminus than at the N-terminus

The helix-coil dynamics of different sections of an alpha-helical model peptide were observed separately by nanosecond temperature jump experiments with IR detection on a series of isotopically labeled peptides. The results show that the helix-coil dynamics of the alpha-helical C-terminus are faster than those of the N-terminus.