Different binding thermodynamics of Ni2+, Cu2+, and Zn2+ to bacitracin A1 determined by capillary electrophoresis

Thermodynamics of the binding of Ni(2+), Cu(2+) and Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni(2+) > Cu(2+) > Zn(2+), with association constant values of (2.3 +/- 0.1)x10(4), (4.9 +/- 0.2)x10(3), and (1.5 +/- 0.1)x10(3) M(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P(0) peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni(2+) and Cu(2+) binding, with a shift of the pK(a) of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group of D-Orn-7 from 9.7 to about 7. Even though on Zn(2+) binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 A lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.     

Metal ions as cofactors for aggregation of therapeutic peptide salmon calcitonin

The effects of multivalent metal ions (Cu(2+)/Zn(2+)/Al(3+)) on the aggregation of salmon calcitonin (sCT)--a therapeutic peptide used worldwide in the treatment of osteoporosis and Paget's disease--have been studied in vitro using NMR (both solution state and solid state), TEM, ThT-fluorescence, and FT-IR spectroscopy. Overall, the various results indicated that the metal-ions-induced conformational transitions in the peptide--mostly toward the β-sheet--facilitate the aggregation of sCT in solution. First, the solution NMR has been used to check the interaction between the peptide and the metal ions. Following this, the formation and characterization of calcitonin aggregates has been performed using TEM, solid state NMR, and FT-IR spectroscopy. The TEM and ThT-fluorescence results revealed that the sCT peptide incubated with Cu(2+) and Zn(2+) metal ions (in aqueous environment) forms globular aggregates, while that with Al(3+) ions forms fibrils. The solid state NMR and FT-IR studies revealed the presence of a substantial amount of β-sheet content in sCT aggregates (formed in the presence of these metal ions) compared to the monomeric sCT, indicating that the metal binding is concomitant with conformational changes. The present study becomes crucial while prescribing this drug peptide under physio-pathological conditions associated with an abnormal accumulation of metal ions (Cu(2+)/Zn(2+)/Al(3+)) in the body (i.e., abnormal metal ion homeostasis).     

Evidence of complete hydrophobic coating of bombesin by trifluoroethanol in aqueous solution: an NMR spectroscopic and molecular dynamics study

Bombesin is a tetradecapeptide that possesses a random coil structure in pure water. In the presence of 30 % (v/v) 2,2,2-trifluoroethanol (TFE), it adopts a partial helical conformation involving the C-terminal amino acids 6-14. This conformational change, known as the TFE effect, is studied here in terms of the solvation state of the peptide at different TFE concentrations by means of intermolecular homo- and heteronuclear NOE measurements. When an aqueous solution of bombesin is titrated with TFE, a continual decrease in the water/peptide interactions and a concomitant increase in the TFE/peptide interactions is observed, and at 30 % (v/v) TFE no homonuclear NOEs between water and the peptide can be detected. The conformational transition of the bombesin molecule is thus accompanied by a complete surface covering with TFE. A parallel molecular dynamics (MD) study of the peptide in aqueous solution with the single-point charge (SPC) water model and in a 30 % (v/v) TFE/water mixture with a recently developed TFE model has also been performed. The 10 ns simulations were in agreement with the experimental data. The calculations indicate stabilisation of the alpha-helix in the H(2)O/TFE mixture, in contrast to the situation in pure water, and clustering of the TFE molecules around the peptide.     

Affinity capillary electrophoresis study of the linkage existing between proton and zinc ion binding to bacitracin A1

Measurements by capillary electrophoresis (CE) of bacitracin A(1) effective mobility at different pH values permitted to estimate the five acidic dissociation constants and the Stokes radii at different protonation stages of the macrocyclic dodecapeptide. The pK(a) values were 3.6 and 4.4 for the two carboxylic groups of the lateral chains of D-Asp-11 and D-Glu-4, respectively, 6.4 for the aza-atom of the imidazole ring of His-10, 7.6 for the amino group of N-terminal Ile-1 and 9.7 for the delta-amino group of D-Orn-7, very close to the values obtained by other researchers by titration experiments. In agreement with a rigid macrocyclic structure the Stokes radii of different protonated forms ranged only between 14.3 and 14.8 A. Best fitting procedures performed on experimental mobility measured at two different pH values (5.50 and 6.72) in the presence of increasing Zn(+2) concentration allowed confirming the model that assumes the binding of Zn(+2) to P(0) peptide form with a 1.5 x 10(3) M(-1) intrinsic association constant. Following to Zn(+2) binding, the pK(a) of the amino group of N-terminal Ile-1 is shifted from 7.6 to 5.9 and the Stokes radius is reduced of about 3 A. The mean charge of the bacitracin A(1)-Zn(+2) complex resulted +1.67 and +1.12 at pH 5.50 and 6.72, respectively. These results suggest that the amino group of N-terminal Ile-1 is not essential for Zn(+2) binding.     

Folding of the 25 residue Abeta(12-36) peptide in TFE/water: temperature-dependent transition from a funneled free-energy landscape to a rugged one

The free-energy landscape of the Alzheimer beta-amyloid peptide Abeta(12-36) in a 40% (v/v) 2,2,2-trifluoroethanol (TFE)/water solution was determined by using multicanonical molecular dynamics simulations. Simulations using this enhanced conformational sampling technique were initiated from a random unfolded polypeptide conformation. Our simulations reliably folded the peptide to the experimental NMR structure, which consists of two linked helices. The shape of the free energy landscape for folding was found to be strongly dependent on temperature: Above 325 K, the overall shape was funnel-like, with the bottom of the funnel coinciding exactly with the NMR structure. Below 325 K, on the other hand, the landscape became increasingly rugged, with the emergence of new conformational clusters connected by low free-energy pathways. Finally, our simulations reveal that water and TFE solvate the polypeptide in different ways: The hydrogen bond formation between TFE and Abeta was enhanced with decreasing temperature, while that between water and Abeta was depressed.     

Diametrically disubstituted cyclam derivative having Hg2+-selective fluoroionophoric behaviors

[reaction: see text] A new fluoroionophore has been synthesized by appending two signaling pyrenylacetamide subunits on the binding motif of 1,8-dimethylcyclam. The designed compound exhibited highly selective and sensitive fluoroionophoric behavior toward Hg(2+) ions of excimer emission in aqueous dioxane (dioxane/H(2)O = 1:9, v/v) solution with a detection limit of 1.3 x 10(-)(6) M. The "ON-OFF" type signaling behavior of the fluoroionophore is due to the metal ion induced conformational changes from folded to open-winged conformations by exploiting the two nearby appended pyrenyl fluorophores.     

Structure and folding of glucagon-like peptide-1-(7–36)-amide in aqueous trifluoroethanol studied by NMR spectroscopy

The conformational changes of free, monomeric glucagon-like peptide-1-(7–36)-amide (GLP-1) in aqueous solution with increasing concentrations of 2,2,2-trifluoroethanol (TFE) were monitored by NMR spectroscopy. It was found that GLP-1 gradually assumes a stable, single-stranded helical structure in water solution when the TFE concentration is increased from 0 to 35% (v/v). No further structural changes were observed at higher TFE concentrations. The structure of GLP-1 in 35% TFE was determined from 292 distance restraints and 44 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The helical structure extends from T7 to K28, with a less well-defined region around G16 and a disordered six-residue N-terminal domain. The folding process of GLP-1 from random coil (in water) to helix (in 35% TFE) is initiated by the formation of the C-terminal segment of the helix that is extended gradually towards the N-terminus of the peptide with increasing concentration of TFE. The exchange rates of the slow exchanging amide protons indicate that the C-terminal part of the helix is more stable than the N-terminal part. Copyright © 2001 John Wiley & Sons, Ltd.     

Evaluation of the metal binding properties of a histidine-rich fusogenic peptide by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) was used to investigate metal ion interactions of the 18 amino acid peptide fragment B18 (LGLLLRHLRHHSNLLANI), derived from the membrane-associated protein bindin. The peptide sequence B18 represents the minimal membrane-binding motif of bindin and resembles a putative fusion peptide. The histidine-rich peptide has been shown to self-associate into distinct supramolecular structures, depending on the presence of Zn(2+) and Cu(2+). We examined the binding of B18 to the metal ions Cu(2+), Zn(2+), Mg(2+), Ca(2+), Mn(2+) and La(3+). For Cu(2+), we compared the metal binding affinities of the wild-type B18 peptide with those of its mutants in which one, two or three histidine residues have been replaced by serines. Upon titration of B18 with Cu(2+) ions, we found sequential binding of two Cu(2+) ions with dissociation constants of approximately 34 and approximately 725 micro M. Mutants of B18, in which one histidine residue is replaced by serine, still exhibit sequential binding of two copper ions with affinities for the first Cu(2+) ion comparable to that of wild-type B18 peptide, but with a greatly reduced affinity for the second Cu(2+) ion in mutants H112S and H113S. For mutants in which two histidines are replaced by serines, the affinity for the first Cu(2+) ion is reduced approximately 3-10 times in comparison with B18. The mutant in which all three histidine residues are replaced by serines exhibits an approximately 14-fold lower binding for the first Cu(2+) ion compared with B18. For the other metal ions under investigation (Zn(2+), Mg(2+), Ca(2+), Mn(2+) and La(3+)), a modest affinity to B18 was detected binding to the peptide in a 1 : 1 stoichiometry. Our results show a high affinity of the wild-type fusogenic peptide B18 for Cu(2+) ions whereas the Zn(2+) affinity was found to be comparable to that of other di- and trivalent metal ions.     

Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate metal ion interactions with salivary peptides histatin 3 (H3) and histatin 5 (H5). Conformational changes of these peptides in the presence of metal ions were studied using circular dichroism spectroscopy. H3 and H5 formed high affinity complexes with Cu(2+) and Ni(2+) and, to a lesser extent, with Zn(2+). Both peptides show the potential for multiple binding sites for Cu(2+) and Ni(2+) and only a single strong binding site for Zn(2+). The binding of a third Cu(2+) ion to H3 seems to enable the binding of a fourth ion to H3. The binding of a second and third Ni(2+) ion to H5 has a similar effect in enabling the binding of a fourth ion. None of the metal ions examined stabilized a regular secondary structure for either peptide. Subtle changes in overall conformation are seen with the addition of Cu(2+) to both H3 and H5.     

Confromational Studies on Peptides Containing Enantiometric α-Methyl α-Amino Acids. Part I. Differential conformational properties of (R)- and (S)-2-methylaspartic acid

The conformational properties of four model peptides of the general formula Ac-Tyr-Xaa-Yaa-Zaa-Ala-Lys-Glu-ala-Ala-Glu-Lys-Ala-Zaa-Yaa-Xaa-Lys-NH₂ (Xaa-Yaa-Zaa = Ala-Ala-(R)-Asp(2-Me), 1 ; Ala-Ala-(S)-Asp(2-Me), 2 ; Ala-Aib-Asp, 3 ; Ala-Ala-Asp, 4 ; Asp(2-Me) = 2-methylaspartic acid; Aib = 2-aminoisobutyric acid) were studied by CD spectroscopy in solution, to evaluate the helix-inducing potential of enantiomerically pure 2-methylaspartic acid as a function of its chirality at C(2). At neutral pH and 1°, all peptides exhibit significant helix formation in aqueous solution, the degree of helicity increasing in the order 4 3 ≈ 1 . Lowering the pH to 2 results in a dramatic increase in helicity for peptide 1 , while the diastereoisomeric peptide 2 now exists in a predominantly unordered conformation. Helix induction by protonated (R)-Asp(2-Me) exceeds Aib-induced helix formation in peptide 3 , and the helix content of 1 in aqueous solution at pH 2 is comparable to the degree of helicity in the strongly helix-inducing solvent 2,2,2-trifluoroethanol.     

NMR, DFT and luminescence studies of the complexation of Zn(II) with 8-hydroxyquinoline-5-sulfonate

Multinuclear ((1)H, (13)C) magnetic resonance spectroscopy, DFT calculations and luminescence techniques have been used to study 8-hydroxyquinoline-5-sulfonate (8-HQS) and its complexes with Zn(ii), in aqueous solution. The study combines the high sensitivity of luminescence techniques, the selectivity of multinuclear NMR spectroscopy with the structural details accessible through DFT calculations, and aims to obtain a detailed understanding of the complexation between the Zn(2+) ion and 8-HQS. In addition to a complete assignment of the (1)H and (13)C NMR signals of 8-HQS, a full speciation study has been performed. Over the concentration region studied, Zn(2+) metal ion forms only one significant complex species with 8-HQS in aqueous solution in the pH range 6-8. Job's method shows that this species has a 1:2 (metal:ligand) stoichiometry. The geometry around the metal centre, according to structural optimization using DFT calculations, is suggested to be square bipyramidal, with two coordinated water molecules mutually trans, and the remaining positions occupied by the donor groups of the two coordinated 8-HQS ligands. On binding to Zn(ii), 8-HQS shows a marked fluorescence compared with the weakly-luminescent free ligand. In addition, as previously noted, there are marked changes in the absorption spectra, which support the use of 8-HQS as a sensitive fluorescent sensor to detect Zn(2+) metal ion in surface waters, biological fluids, etc. Based on results of the structural studies, suggestions are made of ways for enhancing fluorescence sensitivity.     

A highly selective and sensitive in vivo fluorosensor for zinc(II) without cytotoxicity

A highly selective and sensitive fluorescent Zn(2+) sensor, 2,6-bis(2-hydroxy-benzoic acid hydrazide)-4-methylphenol (1), was designed and synthesized. In aqueous THF (4 : 6 v/v) ligand 1 induces a 2 : 1 complex formation with respect to Zn(2+) at physiological pH. This probe features visible light excitation(390 nm) and emission (490 nm) profiles, excellent selectivity responses for Zn(2+)over other competing biological metal ions with K(d) < 1 pM(2), LOD < 1 ng L(-1) and about 680 fold enhancement in fluorescent intensity upon Zn(2+) binding. It also exhibits cell permeability and intracellular Zn(2+) sensing in A375 human melanoma cancer cell.     

Speciation of cyclo(Pro-Gly)<Subscript>3</Subscript> and its divalent metal-ion complexes by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to study the binding of selected group II and divalent transition-metal ions by cyclo(Pro-Gly)3 (CPG3), a model ion carrier peptide. Metal salts (CatXn) were combined with the peptide (M) at a molar ratio of 1:10 M/Cat in aqueous solvents containing 50% vol/vol acetonitrile or methanol and 1 or 10 mM ammonium acetate (NH4Ac). Species detected include [M+H]+, [M+Cat-H]+, [M2+Cat]2+, [M+Cat+Ac]+, and [M+Cat+X]+. The relative stabilities of complexes formed with different cations (Mg2+, Ca2+, Sr2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+) were determined from the abundance of 1:1 and 2:1 M/Cat species relative to that of the unbound peptide. The largest metal ions (Ca2+, Sr2+, and Mn2+) formed the most stable complexes. Reducing the buffer concentration increased the overall extent of metal binding. Results show that the binding specificity of CPG3 depends upon the size of the metal ion and its propensity for electrostatic interaction with oxygen atoms. Product ion tandem mass spectrometry of [M+H]+ and [M+Cu-H]+ confirmed the cyclic structure of the peptide, although the initial site(s) of metal attachment could not be determined.     

Photo-controlled metal-ion (Zn2+ and Cd2+) release in aqueous Tween-20 micelle solution

Photo-controlled metal-ion (Zn(2+) and Cd(2+)) release in aqueous micelle solution (tris-HCl, pH = 7.4) has been described using 2-((2-mercaptophenylimino)methyl) phenol as ligand. It is found that both the ligand-Zn complex (1) and the ligand-Cd complex (2) are stable in micelle solution, and Zn(2+) (Cd(2+)) can be released from the complex with 365 nm light trigger. Accompanying the metal-ion release, the ligand is photo-converted to 2-(benzothiazol-2-yl) phenol (3) as product, and the turn-on fluorescence is detected. The fluorescence intensity increases with the photo-triggered release until Zn(2+) (Cd(2+)) is completely released, which is beneficial for monitoring the process of photo-controlled metal ion release. Control experiments demonstrate that no binding occurs between 3 and Zn(2+) (Cd(2+)) in micelle solution and there is no binding between cations and micelle, either.     

Tandem repeating modular proteins avoid aggregation in single molecule force spectroscopy experiments

We have used single molecule force spectroscopy to explore the unfolding and refolding behavior of the immunoglobulin-like I27 protein in aqueous 2,2,2-trifluoroethanol (TFE). In bulk solution experiments, a 28% v/v TFE solution has previously been observed to enhance intermolecular attractions and lead to misfolding and aggregation of tandem modular proteins of high sequence identity. In our single molecule experiments, however, we measure successful refolding of the polyprotein I27(8) in all TFE solutions up to 35% v/v. Using a single molecule micromanipulation technique, we have shown that refolding of a polyprotein with identical repeats is not hindered by the presence of this cosolvent. These experimental results provide new insight into the properties of tandem repeating proteins and raise interesting questions as to the evolutionary success of such proteins in avoiding misfolding and aggregation.     

Separation and evaluation of soybean protein hydrolysates prepared by immobilized metal ion affinity chromatography with different metal ions

Metal ion affinity chromatography is widely used to purify peptides on the basis of the dissimilarities of their amino acids. However, researchers are interested in the separation differences between different metal ions in this method. In our study, four kinds of commonly used metal ions are compared by the amount of immobilized metal ion on iminodiacetic acid-Sepharose and binding amount of soybean peptide to immobilized iminodiacetic acid-Mn(+) adsorbents and evaluated by high-performance liquid chromatography (HPLC) profiles. The results show that due to the different adsorption behaviors of metal ions, the binding ability order of soybean protein peptide on the column should be Fe(3+) > Cu(2+) > Zn(2+) > Ca(2+). The HPLC profiles show that peptides adsorbed by four kinds of metal ions display similar strong hydrophobic characteristics.     

金属离子对齐多夫定与牛血清白蛋白结合作用的影响

用荧光光谱法和紫外分光光度法研究了水溶液(Tris-HCl缓冲溶液, pH 7.1)中齐多夫定(ZDV)与牛血清白蛋白(BSA)的结合作用及三种金属离子(Cu2+, Mg2+, Zn2+)对其的影响. 结果表明: 齐多夫定及金属离子均导致BSA的内源荧光猝灭, 猝灭机制均为静态猝灭; 齐多夫定与BSA间存在较强结合作用, 热力学参数△H和△S分别为-10.2 kJ·mol-1和77.5 J·mol-1·K-1 (298 K), 表明其结合力以静电作用力为主; 298 K下结合常数、结合位点数和结合距离分别为6.92×105 L·mol-1、1.18和2.28 nm; 温度升高结合常数和结合位点数减小. 三种金属离子均导致ZDV与BSA的结合常数减小, 结合距离增大.     

New dioxadiaza- and trioxadiaza-macrocycles containing one dibenzofuran unit with two amino pendant arms: synthesis, protonation and complexation studies

New dioxadiaza- and trioxadiaza-macrocycles containing one rigid dibenzofuran unit (DBF) and N-(2-aminoethyl) pendant arms were synthesized, N,N'-bis(2-aminoethyl)-[17](DBF)N(2)O(2) (L(1)) and N,N'-bis(2-aminoethyl)-[22](DBF)N(2)O(3) (L(2)), respectively. The binding properties of both macrocycles to metal ions and structural studies of their metal complexes were carried out. The protonation constants of both compounds and the stability constants of their complexes with Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Pb(2+) were determined at 298.2 K, in aqueous solutions, and at ionic strength 0.10 mol dm(-3) in KNO(3). Mononuclear complexes with both ligands were formed, and dinuclear complexes were only found for L(2). The thermodynamic binding affinities of the metal complexes of L(2) are lower than those of L(1) as expected, but the Pb(2+) complexes of both macrocycles exhibit close stability constant values. On the other hand, the binding affinities of Cd(2+) and Pb(2+) for L(1) are very high, when compared to those of Co(2+), Ni(2+) and Zn(2+). These interesting properties were explained by the presence of the rigid DBF moiety in the backbone of the macrocycle and to the special match between the macrocyclic cavity size and the studied larger metal ions. To elucidate the adopted structures of complexes in solution, the nickel(II) and copper(II) complexes with both ligands were further studied by UV-vis-NIR spectroscopy in DMSO-H(2)O 1 : 1 (v/v) solution. The copper(II) complexes were also studied by EPR spectroscopy in the same mixture of solvents. The crystal structure of the copper complex of L(1) was also determined. The copper(II) displays an octahedral geometry, the four nitrogen atoms forming the equatorial plane and two oxygen atoms, one from the DBF unit and the other one from the ether oxygen, in axial positions. One of the ether oxygens of the macrocycle is out of the coordination sphere. Our results led us to suggest that this geometry is also adopted by the Co(2+) to Zn(2+) complexes, and only the larger Cd(2+) and Pb(2+) manage to form complexes with the involvement of all the oxygen atoms of the macrocyclic backbone.     

Coordination geometry of cadmium at the zinc and copper sites of superoxide dismutases: a study using perturbed angular correlation of gamma-rays from excited 111Cd.

111Cd time-differential perturbed gamma-gamma angular correlation (PAC) has been used to investigate the Zn site in yeast and bovine copper and zinc-containing superoxide dismutases by substitution of the zinc ions with excited 111Cd(2+) ions. The PAC spectra obtained from the enzymes in aqueous solution reveal a single coordination geometry of 111Cd(2+) showing that the coordination of 111Cd(2+) to the Zn site in the two subunits is identical. Furthermore, the PAC spectra of the yeast and bovine enzymes show that the Zn sites are very similar in the two enzymes. The PAC experiments show a clear difference depending on whether the copper ion is in the oxidized or the reduced state. In the latter case the results resemble those obtained for derivatives with no metal ion at the Cu site. Hence the coordination geometry of the Zn site in these two situations must be similar, and it is very unlikely that the imidazole ring of His61 bridges the two metal ions in the reduced enzyme. The PAC spectrum of 111Cd(2+) ions at the Zn site with copper(II) ions at the Cu site is in agreement with that predicted by applying the angular overlap model (AOM) to the known crystal structure of the bovine enzyme, with known nuclear quadrupole interactions for the ligands involved. Furthermore results from experiments with copper in the reduced state show that reduction of the copper ion causes a significant change at the Zn site. An explanation for this conformational change has been proposed by computer modelling. The PAC experiments also show that it is possible to incorporate cadmium ions into the Cu site in the absence of copper ions, and the result has also been interpreted in terms of the AOM.