Structural and dipolar properties of the voltage-dependent pore former alamethicin in octanol/dioxane.

Dielectric constant and loss of the membrane-active peptide alamethicin in octanol/dioxane mixtures have been measured at frequencies between 5 kHz and 50 MHz. On the basis of a rotational mechanism of dipolar orientation, the observed dispersion provides information regarding size, shape, and dipole moment of the structural entities which the solute may assume in media of diverse lipophilicity. Particularly detailed results are obtained in a pure octanol solvent where an apparent molecular weight of alamethicin could be determined. It turns out that in this quite lipophilic medium most of the peptide material exists as a monomer particle that has approximate length and diameter of 35 and 13 A, respectively. It carries a dipole moment of approximately 75 Debye units (directed nearly parallel to the long axis). At our concentrations of a few milligrams per milliliters, appreciable formation of dimers by head-to-tail linkage is indicated. When the octanol content is reduced by adding greater amounts of dioxane, larger particles are encountered. This is accompanied by a decrease of the effective polarity. The inherent increase of hydrophilicity in the dioxane-enriched solvent apparently favors another monomer conformation that has a low dipole moment and easily aggregates to some kind of micelle.     

Effect of concentration on the aggregation of poly(γ-benzyl-L-glutamate) in dioxane: Critical concentration and maximum length for linear head to tail aggregates

Dielectric relaxation studies in the frequency range 100 Hz to 2 MHz of poly(γ-benzyl-L -glutamate) in dioxane have been carried out over a range of concentration 10^?4–10^?2g/g. The structure of aggregates is analyzed in terms of dipole moment and relaxation time. A critical concentration (? 10^?3 g/g for the studied molecular weights) has been determined below which the aggregates are found to have linear head to tail type structure. Above the critical concentration a different structure of aggregates is apparent which could not be fully analyzed by these measurements alone. Possible forms of aggregation above the critical concentration are discussed. Formation of long range order which would lead to nematic liquid crystalline phase at higher concentrations has been discussed as one of the possible explanations for the observed behavior above the critical concentration. Maximum length of linear head to tail type aggregates for poly(γ-benzyl-L -glutamate) in dioxane as determined from these results correspond to an α-helix of molecular weight 210,000. A slight difference in the purity of dioxane has been shown to have an influence on the reproducibility of the state of aggregation as well as on the rate of disaggregation on dilution.     

Dielectric probe of the electric-field-sensitive peptide alamethicin

Dielectric constant and loss of alamethicin in solvents of various degrees of lipophilicity (namely mixtures of n-octanol and dioxane) have been measured at frequencies from 5 kHz to 50 MHz. By means of a conventional Cole-Cole approach, dielectric properties were evaluated to obtain information about the structural and dipolar properties of the peptide in view of its function as a voltage-dependent pore former in membranes. The results for a pure octanol solvent (together with an apparent molecular weight determined by ultracentrifugation) indicate the existence of primarily monomeric particles of quite elongated shape and of comparatively high dipole moment. Adding dioxane was found to yield considerable aggregation and a decrease of polarity.     

Structure of aggregates and helix-coil transition of polypeptides by dielectric measurements. I. Poly-gamma-benzyl-L-glutamate in dioxane and dioxane-dichloroacetic acid

Dielectric dispersion measurements on poly-γ-benzyl-L -glutamate (PBLG) in dioxane and dioxane–dichloroacetic acid (DCA) mixtures in the frequency range 200 Hz–2 MHz were made in order to study the structure of molecular aggregates. The structure of aggregates is explained on the basis of the variation of dipole moment and relaxation time with degree of aggregation. PBLG was found to form linear head-to-tail-type aggregates in dioxane. These aggregates gradually reduce in size without loosing their α-helical structure during the process of disaggregation obtained by either adding DCA to the solution in dioxane or by heating. It was confirmed that the addition of 30 wt % DCA completely destroys the aggregation of PBLG in dioxane at 30°C. Thermal disaggregation, however, was not complete even at a temperature approaching the boiling point of the solvent. A reaction scheme for aggregation is proposed and equilibrium constants are calculated at various stages of aggregation. The enthalpy of aggregate formation is found to be ?3 kcal/mol. Results of optical rotatory dispersion measurements of the helix–coil transition in this system are also presented.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

Trichotoxin A40. Purification by counter-current distribution and sequencing of isolated fragments

The isolation of the membrane-modifying polypeptide antibiotics from the mycelium of Trichoderma viride 5242 was optimized via extraction with dichloromethane and chromatography on Sephadex LH-20. The components trichotoxin A40 and A50 were separated from each other and purified by multiplicative counter-current distribution. The sequence of proteinase-resistant trichotoxin A40 was determined by combined gas chromatography and mass spectrometry of three isolated N-acetylated dodecapeptides and two N-prolylhexapeptides obtained after selective trifluoroacetolysis. Including amino acid exchanges due to natural microheterogeneity, the sequence is Ac-Aib-Gly(LAla)-Aib-LLeu-Aib-LGln-Aib-Aib-Aib(LAla )-LAla-Aib-Aib-LPro-LLeu -Aib-DIva(Aib)-LGlu-LValol. In contrast to the eicosapeptide alamethicin, trichotoxin A40 contains only 18 residues, with a higher proportion of alpha-aminoisobutyric acid (Aib), C-terminal L-valinol (Vol), one D-isovaline (Iva) and no proline at the N-terminal part.     

Intermolecular association and molecular weight of aggregates of a monodisperse homohexapeptide,Nps-[Glu(OBzl)]6-NHEt,in solution

Light scattering from the solutions of Nps-[Glu(OBzl)]₆-NHEt in dioxane or ethylene dichloride has been measured at different concentrations, and a critical concentration of intermolecular association is found to exist, which is equal to the critical concentration of β-form formation. The Debye plot of light scattering leads to the molecular weight of aggregates at the critical concentration, which corresponds to an aggregation number 15 in dioxane and 53 in ethylene dichloride. In the latter solvent the aggregates further associate into a larger aggregate consisting of 330 molecules when the concentration is increased beyond the critical concentration. The content of β-form, which is a measure of number of hydrogen bonds, is derived from the ir data previously obtained. The results on the modes of intermolecular association and hydrogen bonding lead to possible structures of aggregates formed by both hydrogen bonds and other nonbonding side-chain interactions.     

Dipole moment of alamethicin as related to voltage-dependent conductance in lipid bilayers.

The dipole moment of alamethicin, which produces voltage-dependent conductance in lipid-bilayer membranes, was measured in mixed solvents of ethanol and dioxane. The value of the dipole moment was found to increase from 40 to 75 DU (Debye units), as the concentration of ethanol increased from 0 (pure dioxane) to 40%. The relaxation frequency of alamethicin also changes from 10 to 40 MHz, depending upon the concentration of ethanol in mixed solvents. The length of alamethicin was calculated by using the relaxation time and was found to range from approximately 40 to 20 A. The dipole moment was independently calculated from voltage-dependent conductance and compared with the measured value. The calculated value was found to be larger than the value of direct measurements, indicating that several alamethicin molecules are required to form a conducting pore and that their dipole moments are oriented parallel to each other.     

Trichotoxin A-40, a new membrane-exciting peptide. Part B. Voltage-dependent pore formation in bilayer lipid membranes and comparison with other alamethicin analogues

Trichotoxin A-40 induces voltage-dependent pores in bilayer lipid membranes comparable to those formed by alamethicin and suzukacillin. The conductance values of the trichotoxin A-40 pores are of similar magnitude and show the same characteristic pattern sequence of non-integral multiples of a unit-conductance step as alamethicin and suzukacillin.However, voltage-jump current-relaxation experiments show significant differences between trichotoxin A-40 and alamethicin and suzukacillin. With trichotoxin A-40 three different relaxation processes could be observed, whereas with alamethicin and suzukacillin only two processes had been distinguished. The fast process in each case is related to pore state transitions and the slower (medium) process to the decay rate of pores. The third very slow process, which is not found with alamethicin and suzukacillin, could not clearly be assigned to a molecular mechanism. Whereas in the case of alamethicin and suzukacillin the relaxation amplitude of the slow process is considerably larger than the relaxation amplitude of the fast process, the reverse is true for trichotoxin A-40, where the largest relaxation amplitude is that of the fast process.Contrary to alamethicin and suzukacillin, trichotoxin A-40 is soluble in the lipid/decane membrane-forming solution, when added from an ethanolic stock solution. Its bilayer-modifying properties are not changed, whether the antibiotic is added to the aqueous salt solution or to the membrane-forming solution.Several different analogues of alamethicin, suzukacillin and trichotoxin A-40 have been investigated and compared with respect to the induced current-voltage characteristics in lipid bilayers. A suzukacillin A-derivative where phenylalaninol had been split off is active as well as trichotoxin A-40 which lacks the phenylalaninol group by nature. Different C-terminal groups like -COOH, -CONH₂, -COOCH₃ and -CO-Ala-Ala-OCH₃ cause qualitative changes but not the loss of the pore-formation property.     

The -conformation and association of low-molecular-weight poly- -benzyl-L-glutamate in solutions

Three samples of poly-γ-benzyl-L -glutamate have been prepared from γ-benzyl-N-carboxy-L -glutamate anhydride with n-hexylamine initiation at anhydride-to-initiator molar ratios, [A]/[I], of 3, 4, and 8, and their conformation and association in ethylene dichloride and dioxane have been investigated by means of infrared spectra and vapor-pressure osmometry. Two conformations, σ-and β-forms, are present in those solvents, and the content of β-form increases with increasing A/I value and concentration. At infinite dilution molecular association is absent, but the number-average molecular weight increases with cocentration, markedly in ethylene dichloride and, to a lesser extent, in dioxane. The fraction of residues involved in associated molecules have been estimated as a function of concentration. Combination of the content of β-structure with the fraction of association leads to the following results. The A/I 3 and 4 polymers form intermolecularly hydrogen-bonded aggregates, in which hydrogen-bonded residues are in the β-structure. The A/I 8 polymer has the intramolecularly hydrogen-bonded β-structure at very low concentrations, but it also forms intermolecularly hydrogen-bonded aggregates at high concentrations.     

Circular dichroism studies of α-aminoisobutyric acid-containing peptides: Chain length and solvent effects in alternating Aib-L-Ala and Aib-L-Val sequences

The CD spectra of the peptides Boc-X-(Aib-X)_n-OMe (n = 1, 2, 3) and Boc-(Aib-X)₅-OMe, where X = L -Ala or L -Val have been examined in several solvents. The X = Ala and Val peptides behave similarly in all solvents, suggesting that the Aib residues dominate the folding preferences of these peptides. The decapeptides adopt helical conformations in methanol and trifluoroethanol, with characteristic negative CD bands at 222 and 205 nm. In the heptapeptides, similar spectra with reduced intensities are observed. Comparison with nmr studies suggest that estimates of helical content in oligopeptides by CD methods may lead to erroneous conclusions. The pentapeptides yield solvent-dependent spectra indicative of conformational perturbations. Peptide association in dioxane results in an unusual spectrum with a single negative band at 210 nm for the decapeptides. Disaggregation is induced by the addition of methanol or water to dioxane solutions. Aggregation of the heptapeptides is less pronounced in dioxane, suggesting that a critical helix length may be necessary to promote association stabilized by helix dipole–dipole interactions.     

A combined spectroscopic and TDDFT investigation of the solute‐solvent interactions of two coumarin derivatives

The UV/Vis absorption and fluorescence characteristics of 3‐cyano‐7‐hydroxycoumarin [ CHC ] and 7‐amino‐4‐methyl‐3‐coumarinylacetic acid [ AMCA‐H ] were studied at room temperature in several neat solvents and binary solvent mixtures of 1,4‐dioxane/acetonitrile. The effects of solvent on the spectral properties are analyzed using single and multi‐parameter solvent polarity scales. Both general solute/solvent interactions and hydrogen bond interactions are operative in these systems. The solvation of CHC and AMCA‐H dyes in 1,4‐dioxane/acetonitrile solvent mixtures has been studied. The solutes CHC and AMCA‐H are preferentially solvated by acetonitrile and a synergistic effect is observed for both molecules in dioxane/acetonitrile solvent mixtures. In addition, using the solvatochromic method the ground‐ and the excited‐state dipole moments of both the dyes were calculated. The ground‐ and excited‐state dipole moments, absorption and emission maxima and HOMO–LUMO gap were also estimated theoretically using B3LYP/6–311+ G (d,p) level of theory in the gaseous phase, dioxane and acetonitrile solvents. Furthermore, changes in dipole moment values were also calculated using the variation of Stokes shift with the molecular–microscopic empirical solvent polarity parameter ( ). The observed excited‐state dipole moments are larger than their ground‐state counterparts, indicating a substantial redistribution of the electron densities in a more dipolar excited state for both coumarins investigated.     

Changes of dipole moments in the series of Boc-(ProLeuGly)n-OBzl oligopeptides and their possible explanation. A preliminary survey

It was found that the dipole moments in the series of Boc-(ProLeuGly)n-OBzl oligopeptides (n = 1-5) measured in dioxane solution demonstrate a distinct concentration dependence beginning from dodecapeptide. This effect may be connected with the appearance of aggregated (triple-helical?) structure in conformation equilibrium. The tendency to minimize the dipole moment of the system may be of some importance in the creation of supercoiled structures of polypeptides.     

Electric and hydrodynamic properties of polypeptides in solution. II. Conformation of poly(L-glutamic acid) in various organic solvents

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol, dimethyl sulfoxide, dimethylformamide, N-methylacetamide, trifluoroacetic acid, dioxane–water mixtures (3:1 and 4:1 by volume), and dioxane–formamide mixture (1:1 by volume) has been measured by the use of the rectangular pulse technique at 30 °C. The intrinsic viscosity has also been measured at the same temperature. The magnitude of the specific Kerr constant and the intrinsic viscosity suggests that PLGA is helical and has a large dipole moment in methanol, dimethyl sulfoxide, dimelhylformamide, N-methylacetamide, and dioxane–water mixtures. In this case we have obtained the length distribution curve and the mean length of PLGA molecules from the decay of the electric birefringence, by applying the method recently developed for helical polypeptides. Furthermore, we have proposed and applied a method of obtaining the mean dipole moment and the optical anisotropy factor from the field strength dependence of the electric birefringence for polydisperse systems on the basis of the knowledge on the length distribution. The results show that PLGA may have a different helical conformation in dimethyl sulfoxide. The specific Kerr constant of PLGA in trifluoroacetic acid is very small, which suggests that PLGA is a random coil in this solvent.     

Octanol blocks fluid secretion by inhibition of capacitative calcium entry in rat mandibular salivary acinar cells

The aliphatic alcohol octanol is thought to modulate enzyme secretion from the exocrine pancreas by the inhibition of gap junction permeability. We have now investigated the effects of octanol on salivary secretion and intracellular calcium concentration ([Ca2+]i), measured in isolated perfused rat mandibular glands and in isolated mandibular acinar cells respectively. Stimulation of perfused glands with 10 microM carbachol (CCh) evoked a rapid increase in fluid secretion followed by a decrease to a sustained elevated level. Application of 1 mM octanol during CCh stimulation inhibited fluid secretion reversibly. In isolated acini, the CCh-induced [Ca2+]i increase was reversibly inhibited by the same concentration of octanol. However, octanol also inhibited the increase in [Ca2+]i in single acinar cells where gap junctions were no longer functional, indicating that octanol directly affected the intracellular Ca2+ signalling pathway. The initial increase in [Ca2+]i induced by 0.5-10 microM CCh, which is due to Ca2+ release from IP3-sensitive Ca2+ stores, was not affected by pretreatment with octanol. In contrast, CCh-, phenylephrine- or thapsigargin-induced Ca2+ entry was almost completely and reversibly inhibited by octanol. Octanol also blocked agonist-evoked Ca2+ entry in pancreatic acinar cells, and thapsigargin-evoked Ca2+ entry in fibroblasts. These data strongly suggest that octanol blocks salivary secretion from mandibular gland by the inhibition of capacitative Ca2+ entry, and raise the possibility that octanol may be a useful tool for inhibiting agonist-evoked Ca2+ entry pathways.     

Structural flexibility of Aib-containing peptides: the N-terminal tripeptide of trichotoxin

The sequence Aib-Gly-Aib which corresponds to the N-terminus of the microheterogeneous peptide antibiotic trichotoxin has been studied crystallographically in the context of different protecting groups. Peptides Ac-Aib-Gly-Aib-OH (A) and Z-Aib-Gly-Aib-OH (B) form beta-turns. Both peptides show a remarkable conformational flexibility forming a large variety of beta-turns of different types.     

Crystal structure of Z-Aib-Aib-Aib-Ala-Ala-Aib-OtBu, a hexapeptide fragment of trichotoxin

The crystal structure of Z-Aib-Aib-Aib-Ala-Ala-Aib-OtBu, an end-protected hexapeptide with a sequence corresponding to residues 7-12 of several trichotoxin A-50 sequence analogues has been determined by X-ray crystallography. The hexapeptide adopts a right-handed 3(10)-helical conformation consisting of four consecutive beta-turns of type III. The helix is stabilized by four intramolecular hydrogen bonds. In the crystal the molecules are connected head to tail with intermolecular hydrogen bonding interactions among translationally related molecules thus forming infinitely long helical columns. The column-column interactions in the crystal are hydrophobic and occur predominantly between antiparallel directed columns.     

Self-association of a high molecular weight subfragment-2 of myosin induced by divalent metal ions

The effect of divalent cations on the self-association of high molecular weight subfragment-2 (long S-2) and low molecular weight subfragment-2 (short S-2) of rabbit skeletal muscle myosin has been investigated. In the presence of millimolar concentrations of Ca2+ or Mg2+ long S-2 associates at neutral pH to form ordered, high molecular weight aggregates whereas short S-2 does not associate. The association process is co-operative and results from binding two to four divalent cations within the light meromyosin-heavy meromyosin (LMM-HMM) hinge region of long S-2. Optical diffraction of electron micrographs of the long S-2 aggregates revealed several periodicities including reflections near 143 A. High molecular weight HMM showed a similar divalent metal induced self-association. Chymotryptic digestion studies of rod filaments reveal that cleavage within the LMM-HMM hinge is also strongly dependent on the presence of divalent cations. At pH 8, in the absence of divalent cations, the S-2 region appears to be displaced away from the filament backbone resulting in rapid proteolysis in the hinge domain. At high cation concentrations (greater than 10 mM) proteolytic cleavage is suppressed. A similar depression of the (substantially lower) hinge cleavage rate was also observed at neutral pH following addition of these divalent metal ions. Results suggest that binding of Mg2+ within the hinge domain under physiological conditions may act to lock the cross-bridge onto the thick filament surface in its resting-state orientation.     

Double coned inverse sandwich complexes [M-(η4-C4H4)-M′] of Gr-IA and Gr-IIA Metals: theoretical study of electronic of structure and second hyperpolarizability

Molecular interaction between dioxane and methanol involves certain polar and nonpolar bonding to form a one to one complex. Interatomic distances between hydrogen and oxygen within 3 Å have been considered as hydrogen bonding. Optimizations of the structures of dioxane-methanol complexes were carried out considering any spatial orientation of a methanol molecule around a chair/boat/twisted-boat conformation of dioxane. From 45 different orientations of dioxane and water, 23 different structures with different local minima were obtained and the structural characteristics like interatomic distances, bond angles, dihedral angles, dipole moment of each complex were discussed. The most stable structure, i.e., with minimum heat of formation is found to have a chair form dioxane, one O-H…O, and two C-H…O hydrogen bonds. In general, the O-H…O hydrogen bonds have an average distance of 1.8 Å while C-H…O bonds have 2.6 Å. The binding energy of the dioxane-methanol complex is found to be a linear function of number of O-H…O and C-H…O bonds, and hydrogen bond length. Graphical Abstract

Sixteen orientations of methanol around dioxane converge to six local minima including the global minima with one H-O…H and two C-H…O hydrogen bonds.     

Electric and flow birefringence properties of myosin in aqueous urea and sodium pyrophosphate.

The electric dipole moment of rabbit skeletal myosin was estimated from the electric and flow birefringence properties. Myosin formed small polydisperse aggregates (0.2-1.1 microM in length) with an apparent electric dipole moment of 5,000-20,000 Debye in aqueous urea or sodium pyrophosphate at low ionic strength. Permanent dipole moment contributed substantially to the apparent dipole moment. An anti-parallel association of myosin was suggested from the dependence of the apparent dipole moment on myosin concentration. Some interactions between myosin and C-protein were detected in 1 M urea by flow birefringence and analytical ultracentrifugation studies. The apparent dipole moment of myosin aggregates was less dependent on myosin concentration in the presence of C-protein.