Fluctuations and the rate-limiting step of peptide-induced membrane leakage.

Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.

Structural features and conformational equilibria of 310-helical peptides in solution by spectroscopic and molecular mechanics studies.

The structural features and conformational equilibria of a series of short, linear Calpha-methylvaline [(alphaMe)Val]-based peptides in methanol were investigated by combining fluorescence resonance energy transfer measurements and molecular mechanics data. IR spectra were employed to determine their secondary structure, which exhibits an intramolecularly H-bonded, 3(10)-helix conformation that is affected by backbone distortions that are enhanced by the shortness of the main chain.

Structural features of model glycopeptides in solution and in membrane phase: a spectroscopic and molecular mechanics investigation.

Model glycopeptides of the general formula Boc-Ala-Thr(G-D)-A(1)-A(2)-Leu-Leu-Lys(N)-Ala-OMe, where D = dansyl (dimethyl aminonaphthalenesulphonyl), G = glucosyl and N = naphthyl, while A(1)-A(2) = Ala-Leu or Aib-Aib, and denoted as D-G-Ala-N and D-G-Aib-N, respectively, were used to investigate glycoprotein-membrane interactions. They carry two fluorophores (D and N), covalently linked to the glucose ring and the lysine side chain, respectively, while the threonine side chain is O-glycosylated. CD spectra in different solvent media suggest that both glycopeptides attain an ordered structure, possibly a helix-like conformation. By combining FRET (fluorescence resonance energy transfer) experiments with molecular mechanics data, the most probable structures of both glycopeptides were built up, starting from both a right-handed (rh) alpha- and 3(10)-helix. They were found to populate an alpha-helical conformation, a result further confirmed by the very good agreement between theoretical and experimental quenching efficiency only observed when the backbone chain was in alpha-helix. The association of D-G-Ala-N with model membranes (liposomes) was studied by CD, fluorescence decay, fluorescence anisotropy, and collisional quenching experiments. The binding does not alter the structural features of the peptide because the CD spectral patterns are unaffected by the association. The peptide orientation inside the phospholipidic bilayer is guided by the polar glucose molecule lying in the water phase. The insertion of the hydrophobic backbone chain into the membrane, seeing the probes only partially accessible from the external solution, is characterized by a significant degree of heterogeneity, an increase in vesicles size, and a relevant stabilizing effect on the membrane itself against rupture by methanol.

Structural features of linear, homo-Aib-based peptides in solution: a spectroscopic and molecular mechanics investigation.

In continuation of our studies on the determination of the structural features of functionalized peptides in solution by combining time-resolved fluorescence data and molecular mechanics results, the conformational properties of a series of linear, homo-Aib peptides in methanol (a structure-supporting solvent) were investigated. These compounds have the general formula P(Aib)nN, where Aib is alpha-aminoisobutyric acid, N is naphthalene and P is the monomethylated protoporphyrin IX, the two latter chromophores being covalently attached to the peptide C- and N-termini, respectively, while n=3, 6 and 9. According to 1H NMR and IR spectra, the peptides investigated largely populate a 3(10)-helical structure in CDCl3, which is also a structure-supporting solvent. Both steady-state and time-resolved fluorescence measurements show a strong quenching of the N emission that parallels an increase of the P fluorescence intensity, suggesting the occurrence of long-range energy transfer from 1N* to ground-state P. Comparison of quenching efficiencies and lifetime pre-exponents with those obtained theoretically from the deepest energy minimum conformers is very satisfactory. The computed structures, built up by partially taking into account the solvent medium, exhibit a rigid, highly compact arrangement, owing to both the 3(10)-helix conformation of the backbone chain and the very few peptide-to-chromophore covalent linkages. As a result, only one or two stable conformations for each peptide were theoretically found, in full agreement with the time-resolved fluorescence data. Orientational effects between the probes must be taken into account for a correct interpretation of the fluorescence decay results, which implies that interconversion among conformational substates of the N linkages is slower than 10 ns, corresponding to the upper limit of the energy transfer characteristic time.

Peptide-sandwiched protoporphyrin compounds mimicking hemoprotein structures in solution.

A series of covalently bound peptide-protoporphyrin-peptide compounds, also carrying naphthalene (N) to allow a photophysical investigation, were synthesized. Their general formula is P(nN)(2), where P refers to protoporphyrin IX, and n to the number of amino acids in the sequence Boc-Leu-Leu-Lys-(Ala)(x) -Leu-Leu-Lys-OtBu of each backbone chain (x = 0-3; n = x + 6). Their structural features in methanol solution were investigated by ir and CD spectra, and by steady-state and time resolved fluorescence experiments as well. The ir spectra indicate that intramolecularly H-bonded conformations form, and CD data in both methanol and water-methanol mixture suggest the presence of alpha-helix structure. Quenching of excited naphthalene takes place by electronic energy transfer from singlet N* to P ground state. Fluorescence decays coupled with molecular mechanics calculations indicate that two conformers for each dimeric peptide are the major contributors to the observed phenomena. These conformers are characterized by a globular, protein-like structure, where the protoporphyrin resides in a central pocket, while the two N groups are externally situated. Of the four N linkages in the two conformers, three of them attain a very similar steric arrangement around the central P molecule, in terms of both center-to-center distance and mutual orientation, while the fourth experiences a different steric disposition as compared to the others. Experimental photophysical parameters satisfactorily compare with those obtained by theoretical calculations, within the Förster mechanism for long-range energy transfer, only when the mutual orientation of the chromophores was also taken into account. This implies that interconversion among conformational substates of probes linkages is slow on the time scale of the energy transfer process.

A spectroscopic and molecular mechanics investigation on a series of AIB-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivative as probes.

Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib)(n) Trp], where n + r = 4, and Toac is a nitroxide spin-labeled C(alpha,alpha)-disubstituted glycine, were investigated by steady-state and time-resolved fluorescence measurements in different solvent media. A related peptide, i.e., cyclo-¿Orn-[(Aib)(2)-Trp-(Aib)(2)-Z]-Asp-[(Aib)(2)-Toac-(Aib)(2)-+ ++OtBu ]¿ [T-cyclo-Trp], was also studied by the same techniques. It is a L-Orn, L-Asp diketopiperazine template, to which two Aib-based chains are covalently attached, each one containing one chromophore only, i.e., Trp or Toac. Whatever the solvent, in the former series of peptides quenching of the excited Trp exhibits three lifetime components and proceeds on a time scale from subnanoseconds to a few nanoseconds, while in the case of the template the same process occurs entirely on the nanoscale time scale, exhibiting two lifetimes only. The ir absorption spectral patterns suggest that the backbone of the peptides examined is in the 3(10)-helical conformation, as earlier determined by x-ray diffraction for T(Aib)(3)Trp in the crystal state. In all cases, the fluorescence results are satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited Trp to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process, allowing us to estimate the dynamics of the process. Molecular mechanics calculations coupled with time decay data made it possible to build up the most probable structures of these peptides in solution.

Structural features of linear (alphaMe)Val-based peptides in solution by photophysical and theoretical conformational studies.

In continuation of our studies on the determination of the structural features of functionalized peptides in solution by combining time-resolved fluorescence data and molecular mechanics results, the conformational features of a series of linear, L-(alphaMe)Val-based peptides have been investigated in methanol. These foldamers have the general formula F[(alphaMe)Val](r)-T-[(alphaMe)Val](2)NHtBu, where (alphaMe)Val = C(alpha)-methylvaline and r = 0-3, while F [= fluoren-9-ylmethoxycarbonyl (Fmoc)] and T [= 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-carboxylic (Toac)] are a fluorophoric N(alpha)-protecting group and a nitroxide-based alpha-amino acid quencher, respectively. According to ir and CD spectra, the longest term of the series (r = 3) attains a 3(10)-helical structure, while the other peptides populate an intramolecularly H-bonded, 3(10)-helix-like conformation affected by dynamic helical distortions, which are enhanced by the shortness of the backbone chain. Such distortions are reflected in both the energy of the stretching mode and the molar extinction coefficient of the H-bonded N-H groups, the former being higher and the latter smaller than those of a stable 3(10)-helix. Steady-state and time-resolved fluorescence measurements in methanol show a strong quenching of Fmoc by the Toac residue, located at different helix positions, depending on the r value. Comparison of quenching efficiencies and lifetime preexponents with those theoretically obtained from the deepest energy minimum conformers, assuming a Förster mechanism, is satisfactory. The computed structures exhibit a rather compact arrangement, which accounts for the few sterically favored conformations for each peptide, in full agreement with the time-resolved fluorescence data. Orientational effects between the probes must be taken into account for a correct interpretation of the fluorescence decay results, implying that interconversion among conformational substates involving the probes is slower than the energy transfer rate.

A spectroscopic and molecular modeling study on novel pseudopeptides exhibiting biological activity.

A series of pseudopeptides, containing two fluorophores, such as naphthalene (N) and indole (I), and exhibiting interesting biological activity as tachykinin receptor antagonists, were investigated by electronic absorption, CD and steady-state fluorescence experiments. In polar solvents (e.g. methanol), bioactivity is coupled with a stacked, charge-separated complex between I and N, the amount of which depends on the stereochemical features and conformational mobility of the central scaffold in the molecules examined. This agrees with the idea that dipolar charged, spatially close, aromatic moieties are important topochemical elements in the mechanism of action of these receptor antagonists. Molecular mechanics calculations allowed us to build up hypothetical, low-energy conformations of a few representative pseudopeptides, whose structural features are consistent with the experimental findings.

Absorption, emission, and chiroptical spectra of neurokinin 1 tachykinin receptor antagonists: the role of charge-transfer states on the biological activity.

A spectroscopic investigation, based on both electronic absorption and emission spectra as well as on chiroptical data, was performed on novel neurokinin 1 (NK1) tachykinin receptor antagonists, exhibiting interesting biological activity. These pseudopeptides have two fluorophores, i.e. indole (I) and naphthalene (N), and a central scaffold with different conformational mobility. Absorption spectra in methanol show the presence of a new band with respect to the sum spectrum of the isolated chromophores at around 285 nm, the intensity of which linearly increases as the bioactivity increases. This absorption disappears by using dioxane as solvent. It is ascribed to an intramolecular I-N charge-transfer (CT) complex that forms to different extent, depending on the flexibility of the scaffold. Under this condition, the molecules fold and apparently attain the correct conformation for competing substance P binding to the NK1 receptor, lending plausibility to the role of dipolar charged, spatially close aromatic moieties as topochemical elements in the mechanism of action of substance P antagonists. The excited-behavior parallels that in the ground state, as the quenching of the singlet state at 340 nm is found to be linearly dependent on the biological activity, too. Upon decreasing solvent polarity (methanol vs dioxane) the emissions of the dipolar state at around 370 nm disappears, while exciplex emission in the range of 400-500 nm occurs. This transition from charge-separated to exciplex-like states by lowering the dielectric constant of the medium very likely reflects a change in the structural features of the intramolecular I-N stacked complex, from a twisted or an asymmetrically overlapped conformation of the indolyl and naphthyl rings to a face-to-face geometry. Implications of the rigidity of the molecules, arising from the formation of the intramolecular CT complex, on the ellipticity are briefly discussed.

Intramolecular electronic energy transfer in peptides carrying naphthalene and protoporphyrin molecules: a spectroscopic and conformational statistics investigation.

Short linear peptides, carrying an AA spacer in the backbone chain (AA = Aib or Ala), and naphthalene (N) and protoporphyrin IX (P) covalently bound to epsilon-amino groups of lysine side chains, were synthesized. The general formula is Boc-Leu-Leu-Lys(P)-(AA)n-Leu-Leu-Lys(N)-OtBu, with n = 0-2. The photophysical behavior of these compounds was investigated in water/methanol 75/25 (v/v) solution by steady-state and time-resolved fluorescence experiments. Quenching of excited naphthyl chromophore takes place by electronic energy transfer to the porphyrin ground state, and proceeds on a time scale of 3-8 ns, while a minor and slower (approximately 45 ns) fluorescence lifetime measures the decay of the exciplexes. The results were compared with those earlier obtained with the P(Ala)nN peptides (n = 0-4) in methanol solution, showing that addition of water does not significantly alter the dynamic relaxation behavior of the systems investigated, but affects the dissipation mechanism of the energy transferred to P. Quenching efficiencies from both fluorescence intensity and fluorescence lifetime measurements follow a different trend as the number of AA units increases, depending on whether AA = Aib or Ala, indicating that there are differences in the structural features of the two series of peptides. Consistently, CD spectral results suggest that the former compounds attain ordered conformations, possibly of the 3(10)-helical type, while the latter populate alpha-helical structures to an extent depending on the chain length. The ir data in dilute CD3OD or CDCl3 solution confirm this conclusion in that there is an increased percentage of intramolecular H bonds in the P(Aib)nN as compared to the corresponding P(Ala)nN peptides. The photophysical results can be well described by a long-range dipole-dipole interaction model, provided the separation distances distribution and mutual orientation of N and P groups are taken into account. The need of using the angular relationships between the probes implies that interconversion among conformational substates of chromophores linkages is slow on the time scale of the transfer process, very likely because of both the amide bond in the linkages and the bulkiness of the donor-acceptor pair.

Association complexes between Fe(III) or Cu(II) ions and chitosan derivatives. A thermodynamic and spectroscopic investigation.

Association complexes between iron(III) or copper(II) ions and deoxylactit-1-yl (1), 2-substituted pentanedioic acid (2), or 2-substituted propanoic acid (3) derivatives of chitosan were prepared and characterized by thermodynamic and spectroscopic measurements. Complex solutions did not show any precipitate or even opalescence, owing to the hydrolysis of free metal ions, within a wide range of [Me(n+)]/[P] molar ratio, even at a pH as high as 10.5 (Me(n+) = Fe3+ or Cu2+). Both equilibrium dialysis and Job plot experiments suggest that the functional groups in each monomeric residue are an effective site of binding for one metal ion. Reduction potentials, as obtained by cyclic voltammetric measurements, indicate that (i) coordination of the aforementioned polymeric ligands to Cu2+ ions stabilizes the oxidized species, and (ii) iron complexes have an oxidation power definitely higher than that of the corresponding copper compounds. Electron paramagnetic resonances (100 or 6 K) and Mössbauer (r.t.) spectra suggest that the order of increasing distortion from idealized geometry is Me(n+)-chitosan approximately Me(n+)-(3) < Me(n+)-(2) < or = Me(n+)-(1). These results are discussed briefly in the light of a few general considerations concerning the structural features of association complexes between macromolecules and transition metal ions.

Copper complexes immobilized to chitosan.

Polymeric ligands, such as 2-substituted pentanedioic acid (2), 2-substituted propanoic acid (3), and deoxylactit-1-yl (4) derivatives of chitosan (1), were used to prepare copper complexes that are widely soluble in aqueous solution. EPR results (100 K) show that all association complexes basically have a tetragonal symmetry. Visible CD spectra suggest, however, that the order of increasing departure from this geometry is Cu-(1) approximately Cu-(3) less than Cu-(2) less than or equal to Cu-(4), the lack of sterically constraining side-chains in (1) and (3) allowing a more symmetric arrangement of ligands around the central metal ion. Results on the catalytic activity of the association complexes for air oxidation of catechol derivatives are also presented.

Branched-chain analogues of linear polysaccharides: a spectroscopic and conformational investigation of chitosan derivatives.

The solution properties and conformational features of 2-substituted propanoic acid (I) and 2-substituted pentanedioic acid (II) derivatives of chitosan were investigated over a wide range of pH by potentiometric, optical and chiroptical measurements, and by theoretical conformational analysis. No significant change is observed in the solution properties of I upon pH variations, in agreement with computational results showing that the conformational features of the polymer do not vary with respect to the charge state of the ionizable groups. In contrast, spectroscopic titration and preliminary 1H-n.m.r. data indicate that conformational equilibria in II are pH-dependent. Consistently, computed models show that both the charge state of the ionizable groups and the chirality of the carbon atom in the side chain control the structural features of the polymer.

Stereoselective electron transfer between chiral substrates and metal chelates anchored to polypeptides.

Electron transfer from ortho-dihydroxy substrates, such as L(+)-ascorbic acid, L-adrenaline, and L-dopa, to iron(III) in [Fe(tetpy)(OH)2]+ ions anchored to sodium poly(L-glutamate) (FeTL) or poly(D-glutamate) (FeTD) was found to proceed stereoselectively when structurally ordered and partially shielded active sites prevent easy approach for redox partner. Oxidant-reductant interactions are then mediated by the polypeptide, whose conformational asymmetry ensures an efficient sterically discriminating environment. Evidence is produced that stereoselectivity chiefly arises from transition state effects, while thermodynamic discrimination is of minor importance. Theoretical models of the diastereomeric electron-transfer complexes were constructed by conformational energy calculations based on Coulombic, nonbonded, and hydrogen-bonded energy terms. The molecular parameters of the models enabled "differential" thermodynamic functions of the diastereomeric pairs and stereoselectivity to be evaluated and satisfactorily compared with those experimentally determined. The models give good insight into the observed topochemical phenomena and support the idea that stereoselectivity is coupled with a remote attack mechanism on the central metal ion where the peripheral tetpy ligand of the active sites acts as an electron-transfer agent.

Cooperative effects in the binding of pyridoxal 5'-phosphate to mitochondrial apo-aspartate aminotransferase.

Titrations of mitochondrial apo-aspartate aminotransferase with pyridoxal 5'-phosphate in the presence of AMP, contrary to what has been observed in the case of the cytosolic isoenzyme [(1983) FEBS Lett. 153, 98-102], show sigmoidal isotherms, with Hill coefficients ranging from nH = 1.4, in the absence of AMP, to nH = 1.8, in the presence of 5.9 mM AMP. The experimental data were successfully fitted by the Monod-Wyman- Changeaux model. The best fit, in the absence of AMP, was obtained with L = 30, KR = 4.72 X 10(-7) M and KT = 1.18 X 10(-5) M. Binding curves in the presence of AMP fit the model by keeping KR as a constant. This implies that AMP could bind to the apoenzyme only in the T state. In contrast, binding curves in the presence of phosphate ion (Pi) showed a less pronounced cooperativity, the Hill coefficient dropping to nH = 1.0 in the presence of 0.1 mM Pi. The above results suggest a regulatory role of AMP and Pi in the reconstitution of aspartate aminotransferase.

Room-temperature magnetic properties of oxy- and carbonmonoxyhemoglobin.

The magnetic susceptibility and the density of human oxy-(HbO(2)) and carbonmonoxyhemoglobin (HbCO) solutions of various concentrations have been measured at room temperature, with pure water used as a calibrant. Solutions of unstripped and stripped HbO(2) at pH 7.2 in unbuffered water solvent were always found to be less diamagnetic than pure water, whereas solutions of HbCO in identical conditions were always found to be more diamagnetic than pure water. After correcting for concentration-dependent density changes and assuming the HbCO samples to be fully diamagnetic, the paramagnetic reduction of the diamagnetic susceptibility of HbO(2) corresponds to a molar susceptibility per heme (chi(M) (heme)) of 2460 +/- 600 x 10(-6) cgs/mol.

Magnetic properties of oxyhemoglobin.

When the magnetic susceptibility of frozen aqueous solutions of human oxyhemoglobin was measured in the range between 25 and 250 K, it showed a temperature-dependent behavior typical of a thermal equilibrium between a ground singlet state and an excited triplet state for two electrons per heme, the energy separation being [2J] = 146 cm-1. By contrast, within the same temperature range, carboxyhemoglobin was found to be diamagnetic, as already reported.