Multiple solution conformations and internal rotations of the decapeptide gramicidin S.

The conformations of every C alpha H-C beta H2 moiety of the peptide gramicidin S are reported. Internal rotation occurs, but distinct preferences for one side chain rotamer, greater than 80%, are found for the D-phenylalanine and ornithine residues. Leucine and valine exhibit more extensive averaging while proline is shown to be at least 90% in the Ramachandran B conformation. The data are consistent with the coexistence of many tertiary conformations of gramicidin S; the statistical weights of the twelve major tertiary conformations consistent with the rotamer populations are reported. The relative statistical weights of the tertiary conformers depend upon temperature and solvent. A comparison of the conclusions from this publication and conformations derived by energy minimization procedures is made. Partial agreement was found, but the calculations have not yet predicted the wealth of coexisting tertiary conformations nor accounted for the subtle effects of solvent. It is proposed that a more complete picture of the conformational dynamics of gramicidin S and other peptides will result from calculations which use as a basis the extensive data reported here.     

The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.     

The effect of the bilayer phase transition on the carbonyl carbon-13 chemical shift anisotropy

Proton enhanced (PE), natural abundance carbon-13 magnetic resonance spectra have been obtained of the carbonyl groups in hydrated dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. A four-fold change in the overall linewidth results on passing from the fluid to crystalline phase. The carbonyl resonance provides a sensitive measure of the changes in mobility experienced by the lipid molecule above and below the phase transition temperature. The spectral shapes derived from both the fluid (T = 45°C) and crystalline (T = 15°C) phases indicate that even in the crystalline phase sufficient molecular motion is present to average the chemical shielding tensor. It is suggested that this motion in the L_β′ phase is a result of dislocations and packing faults diffusing in the plane of the bilayer. Because of the small size of the chemical shielding interaction (approx. 3 kHz for ω₀ = 22.63 MHz) lipid diffusion coefficients of order 10^?10 cm²/sec observed in the L_β′ phase [1] are effective in averaging the shielding tensor.A comparison is made with the perturbation suffered by the carbonyl groups in the L_α phase in the presence of substantial amounts of cholesterol or the polypeptide gramicidin A.     

4,4'-(Z)-dehydrophenylalanine]gramicidin S with stabilized bioactive conformation and strong antimicrobial activity

Dehydrophenylalanine (delta Phe) was incorporated into an antibiotic peptide gramicidin S (GS) in place of D-Phe4,4' to prepare an unsaturated analog. Conformational analysis with 1H-NMR indicated that the unsaturated analog has much the same backbone conformation as that of natural gramicidin S as shown by NOE experiments. Studies on temperature dependences and on the chemical shift differences showed that the hydrogen bonds between Val-NH and Leu-CO in the unsaturated analog are strengthened by the incorporation of delta Phe4,4'. This resulted in the reinforcement of the beta-sheet structure which is the most important structural element for GS bioactivity. [delta Phe4,4']gramicidin S exhibited indeed very strong antimicrobial activities against Gram-positive bacteria as well as the natural peptide.     

The conformation of linear gramicidin is sequence dependent

A comparative monolayer and infrared study of analogues of gramicidin A containing either tyrosines or naphthylalanines instead of tryptophans indicates that the nature of the aromatic residues influences the favoured conformation of the peptides. Polar residues favour the single stranded _DL helix while non polar residues favour the double stranded helix. For partly tryptophan to naphthylalanine substituted analogues the positions of the substitutions orientate the favored conformation. The nature of these substitutions may also modify the peptide-lipid interactions. Correspondence to: F. Heitz Chemical structures of the gramicidin A analogues mentioned in this paper. The differences from gramicidin A are underlined. GM^–: GT:     

Conformation and Orientation of Gramicidin a in Oriented Phospholipid Bilayers Measured by Solid State Carbon-13 NMR

Three analogues of the helical ionophore gramicidin A have been synthesized with ¹³C-labeled carbonyls (¹³C=O) incorporated at either Gly², Ala³, or Val⁷. A fourth compound incorporated ¹³C at both the carbonyl and α-carbon of Gly² within the same molecule. These labels were studied using solid-state, proton-enhanced, ¹³C nuclear magnetic resonance (NMR) in hydrated dispersions of dimyristoylphosphatidylcholine (DMPC)-gramicidin A. The dispersions were aligned on glass coverslips whose orientation to the magnetic field could be varied through 180°. The orientation dependence of the NMR spectrum was used to obtain an accurate measurement of the ¹³C chemical shift anisotropy (CSA), and in the case of the fourth compound, the ¹³C—¹³C dipolar coupling constant. From the measured CSA and estimates of the orientation of the ¹³C shielding tensor, we are able to determine the direction of the ¹³C=O bonds and to compare these with the predictions of the various reported models for the configuration of gramicidin A in phospholipid bilayers. Our results are consistent with the left-handed ππ^6.3_LD single-stranded helix (Urry, D. W., J. T. Walker, and T. L. Trapane. 1982. J. Membr. Biol. 69:225-231). The right-handed ππ^6.3_LD single-stranded helix observed for gramicidin A in sodium dodecyl sulfate micelles (Arseniev, A. S., I. L. Barsukov, V. F. Bystrov, A. L. Loize, and Yu A. Ovchinnikov. 1985. FEBS (Fed. Eur. Biochem. Soc.) Lett. 186:168-174) yields a poorer fit to the data. However, the width of the carbonyl resonances suggests a distribution of molecular geometries possibly resulting from a spread in the helix pitch and handedness. Double-stranded helices and β sheet structures are excluded. In dispersions in which the lipid is in the L_α phase, the gramicidin A undergoes rapid reorientation about an axis which is centered on the normal to the plane of the coverslips. When the supporting lipid is in the L_β′ phase the helices are rigid on the timescale of ¹³C-NMR. The configuration of gramicidin A is unaltered by L_α-L_β′ phase transition of the bilayer lipid.     

NMR studies on angiotensin II: Histidine and phenylalanine ring stacking and biological activity

The conformations of angiotensin II and the antagonist [Sar¹, Ile⁸]angiotensin II in dimethylsulfoxide have been examined by high resolution proton magnetic resonance spectroscopy at 400MHz. The chemical shifts for the aromatic protons of the phenylalanine residue in angiotensin II are consistent with shielding and restricted rotation for this side-chain. The chemical shifts for the histidine C₂ and C₄ protons in angiotensin II also indicate shielding, whereas these same protons in the antagonist [Sar¹, Ile⁸]angiotensin II do not demonstrate this shielding influence. These findings suggest a stacking interaction for the histidine and phenylalanine side-chains in angiotensin II which is important for activating angiotensin receptors.     

Computationally designed β‐turn foldamers of γ‐peptides based on 2‐(aminomethyl)cyclohexanecarboxylic acid

The γ‐peptide β‐turn structures have been designed computationally by the combination of chirospecific γ 2 , 3 ‐residues of 2‐(aminomethyl)cyclohexanecarboxylic acid (γAmc₆) with a cyclohexyl constraint on the C^α?C^β bond using density functional methods in water. The chirospecific γAmc₆ dipeptide with the (2S,3S)‐(2R,3R) configurations forms a stable turn structure in water, resembling a type II′ turn of α‐peptides, which can be used as a β‐turn motif in β‐hairpins of Ala‐based α‐peptides. The γAmc₆ dipeptide with homochiral (2S,3S)‐(2S,3S) configurations but different cyclohexyl puckerings shows the capability to be incorporated into one of two β‐turn motifs of gramicidin S. The overall structure of this gramicidin S analogue is quite similar to the native gramicidin S with the same patterns and geometries of hydrogen bonds. Our calculated results and the recently observed results may imply the wider applicability of chirospecific γ‐peptides with a cyclohexyl constraint on the backbone to form various peptide foldamers. © 2012 Wiley Periodicals, Inc. Biopolymers 97:1018–1025, 2012.     

Use of natural-abundance 15N-nmr spectroscopy to investigate the secondary structure of peptides: Gramicidin S

The natural-abundance ¹⁵N-nuclear magnetic resonance (nmr) spectrum of the cyclic decapeptide gramicidin S has been measured and assigned in the solvents dimethyl sulfoxide, methanol, and 2,2,2-trifluoroethanol. Three methods have been investigated to distinguish between peptide groups which are exposed to or shielded from the solvent. The solvent dependence of the ¹⁵N chemical shift is correlated with the two types of peptide group in gramicidin S? those with the carbonyl group exposed or shielded from the solvent. The second method monitors the lability of the N? H proton (via the collapse of the reduced ¹⁵N-¹H coupling) in the presence of added base used to promote intermolecular exchange—peptide protons shielded from the solvent exchange more slowly. The third method looks at the temperature dependence of the ¹⁵N chemical shifts in dimethyl sulfoxide. Here the data are not so distinctive as to allow the differentiation between solvent-exposed or shielded N? H bonds at all peptide groups.     

Conformational and spectral analysis of the polypeptide antibiotic N-methylleucine gramicidin S dihydrochloride by nuclear magnetic resonance.

The 220-MHz proton magnetic resonance spectrum of the cyclic decapeptide antibiotic, mono-N-methylleucine gramicidin S, is reported and all the resonances have been assigned to specific protons of the constituent amino acids. Three methods--temperature dependence and solvent mixture (methanol-trifluoroethanol and dimethyl sulfoxide-trifluoroethanol) dependence of peptide NH proton chemical shifts and proton deuteron exchange--habe been utilized to delineate peptide NH protons. The results of the above methods, coupled with the observed vicinal alpha-CH-NH coupling constants and chemical shifts, indicate that in trifluoroethanol the peptide NH PROTONS OF D-Phe4, D-Phe9, L-Orn2, and L-Val6 are exposed to the sovent, and those of L-Val1, L-Orn7, and L-Leu8 are solvent shielded and intramolecularly hydrogen bonded. In trifluoroethanol, dimethyl sulfoxide, and methanol, the decapeptide has no C2 symmetry, and there are only minor conformational differences in the different solvents. In the proposed conformation in trifluoroethanol, one-half of the decapeptide retained the hydrogen bonding pattern of gramicidin S, i.e. cyclo-(L-Val1 NH--O-C L-Leu8) (a beta turn) and cyclo-(L-Leu8 NH--O-C L-Val1). The second half of the molecule exhibits a different type of stable beta turn involving the ten-atom hydrogen-bonded ring, cyclo-(L-Orn7-NH--O-C D-PHE4).     

Membrane-bound structure and alignment of the antimicrobial β-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR

The antimicrobial properties of the cyclic -sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we present the membrane-bound structure and alignment of a derivative of this peptide, based on angular and distance constraints. Solid-state ¹⁹F-NMR was used to study a ¹⁹F-labelled gramicidin S analogue in dimyristoylphosphatidylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivalent leucine side chains were replaced by the non-natural amino acid 4F-phenylglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence, the distance between the two ¹⁹F-labels was measured from their homonuclear dipolar coupling as 6 Å, in good agreement with the known backbone structure of natural gramicidin S in solution. By analyzing the anisotropic chemical shift of the ¹⁹F-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and described its temperature-dependent mobility. In the gel phase, the ¹⁹F-labelled gramicidin S is aligned symmetrically with respect to the membrane normal, i.e., with its cyclic -sheet backbone lying flat in the plane of the bilayer, which is fully consistent with its amphiphilic character. Upon raising the temperature to the liquid crystalline state, a considerable narrowing of the ¹⁹F-NMR chemical shift dispersion is observed, which is attributed the onset of global rotation of the peptide and further wobbling motions. This study demonstrates the potential of the ¹⁹F nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times.     

Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase

The epimerase (E) domain of the three-domain (ATE) initiation module of Bacillus brevis gramicidin S synthetase equilibrates the Calpha configuration of the phenylalanyl moiety presented as Phe-S-4'-phosphopantetheine-modified (Ppant) acyl enzyme. Mutants at 22 residues of this E domain that are conserved across the approximately 450 residue E domains of nonribosomal peptide synthetases were constructed, and the PheATE derivatives expressed in Escherichia coli as C-terminal His tag fusions and then purified and assayed for three activities: (1) the L-Phe Calpha-[(3)H] exchange to solvent, (2) the rate of approach to D-Phe/L-Phe-S-Ppant acyl enzyme equilibrium from either L- or D-Phe, and (3) the rate of Phe-Pro dipeptidyl-S-Ppant enzyme formation with the downstream ProCAT module. We found that for wild-type PheATE epimerization is much faster than subsequent condensation, leading to a 1.9:1 ratio of D-Phe-S-Ppant/L-Phe-S-Ppant acyl enzyme. Only D-Phe is then transferred to yield D-Phe-L-Pro-S-Ppant ProCAT acyl enzyme. Among the mutants generated, three PheATE constructs, H753A, D757S, and Y976A, showed no detectable Calpha-(3)H washout, while E892A and R896A were among a larger set partially impaired. All these mutants were dramatically impaired in approach to D-Phe/L-Phe-S-Ppant equilibrium from either D- or L-Phe, while another construct, D767S, was asymmetrically impaired only for D-to-L-Phe direction. In the D-Phe-L-Pro dipeptidyl-S-Ppant condensation assay, the H753A and E892A forms of PheATE were only slightly active from L-Phe but unimpaired from D-Phe; N975A epimerizes faster than Y976A from L-Phe. When the chirality of the Phe-Pro-diketopiperazine released product was analyzed the D,L/L,L ratio from wild-type PheATE and ProCAT was 98:2. From E892A and N975A it was comparably 95:5 and 92:8, but H753A and Y976A yielded 56% of the L,L-product, reflecting a gain of function to transfer L-Phe. The 98:2 preference of wild-type PheATE for D-Phe transfer reflects the kinetically controlled stereopreference of the condensation (C) domain of ProCAT for the D-Phe-S-Ppant donor substrate. It may be that other NRPS C domains immediately downstream of E domains will likewise be D-selective.     

The structure of the gramicidin A transmembrane channel

Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11. Simultaneous conductance and fluorescence measurements on planar lipid bi-layer membranes containing dansyl gramicidin C yielded four conclusions: (1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 ± 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. (2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel. (3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 Å-hydrocarbon thickness than in a 47 Å-glycerolester membrane. The dimerization constant in a 48 Å-phosphatidyl choline membrane was 200 times larger than in a 47 Å-glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. (4) We were readily able to detect 10^?14 mole cm^?2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square μm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes. An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazo-benzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a² and d² were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a tetramer channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. The various models which have been proposed for the conformation of the gramicidin transmembrane channel are briefly discussed.     

Rotation of a single swollen thylakoid vesicle in a rotating electric field. Electrical properties of the photosynthetic membrane and their modification by ionophores,lipophilic ions and pH

Rotation of single swollen thylakoid vesicles (‘blebs’) was induced by means of a rotating electric field of strength 104 V · cm⁻¹, inducing a membrane voltage of 72 mV peak. Within the range of medium conductives described (40–300 μS · cm⁻¹), measurement of the field frequency (2–100 kHz) giving maximum rotation rate is equivalent to measuring the electrical time constant of the bleb membrane. Hence the membrane capacity (specific capacitance) was determined, and the value found at pH 8.1 (0.93 ± 0.07 μF · cm⁻²) is in agreement with values deduced from measurements using other techniques. However, the capacity was also found to decreased with pH: a minimum value of 0.77 ± 0.01 μF · cm⁻² was measured at pH 4.4. The present study was extended to measurements of the effects of the lipid-soluble anion of dipicrylamine on the membrane capacity. At pH 7.2 and dipicrylamine concentration of 1.0 μM, a minimum estimate of the apparent membrane capacity was found to be 2.0 ± 0.2 μF · cm⁻², with 2.6 ± 0.2 μF · cm⁻² being observed at 5.0 μM concentration. In addition, it was found possible to measure the membrane resistivity (specific resistance) in the presence of either gramicidin (1.0 to 10 nM) or valinomycin (1.0 to 10 μM). In the case of gramicidin, it was possible to derive a maximum estimate of the mean channel conductance, and this agrees very well with the values for individual, single channels that may be deduced from artificial bilayer work. Unless the gramicidin channels in blebs are in fact substantially more conductive than in artificial bilayers, this indicates that a high percentage of the added gramicidin forms channels which are open for most of the time. In the case of valinomycin, a much greater amount had to be added to produce the same reduction of membrane resistivity as seen with a given concentration of gramicidin. However, calculations indicate that the majority of this effect is due to the difference in partioning behaviour of the two ionophores.     

Solution, interfacial, and membrane properties of gramicidin A.

On the basis of the calculated magnitude of the unidirectional flux through a gramicidin channel, it was predicted that a single conducting event should be sufficient to release trapped ²²Na⁺ or ⁴²K⁺ from phospholipid vesicles with a consequent apparent loss of $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ ion selectivity. In support of this prediction, the introduction of gramicidin to the bathing solution of phospholipid vesicles containing trapped ²²Na⁺ or ⁴²K⁺ led to a release of vesicle contents which was consistent with the expectation that, for each gramicidin dimer present, the contents of approximately one vesicle are released. The predicted apparent loss of $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ selectivity was also observed. Evidence was also presented suggesting some movement of gramicidin from vesicle to vesicle. The fluorescent intensity of gramicidin decreases with time when added to aqueous solutions at very low concentrations. It is proposed that this is a consequence of the extremely low solubility of gramicidin in water. On the basis of area per molecule calculations at the air-water interface, it was argued that the most likely conformation of gramicidin existing at the air-water interface, of those proposed in the literature, was that of Π_L,D⁶ helix.     

Binding of thallium and other cations to the gramicidin A channel. Equilibrium dialysis study of gramicidin in phosphatidylcholine vesicles

Gramicidin A is a linear peptide antibiotic which forms dimer transmembrane channels selective for small monovalent cations, including thallium ions (Tl⁺) which are strongly bound. While there is great interest in the number of ion-binding sites per channel and the affinities of the sites for the various cations, measurements of the kinetics of ion permeation yield these equilibrium parameters only as indirect estimates dependent on the model assumed for the channel. Sonicated lipid vesicles. containing 1 mole of gramicidin per 30 moles of dimyristoylphosphatidylcholine. can be prepared with 5 mm-gramicidin. Evidence from our previous spectroscopic studies strongly supports the belief that this gramicidin is in the form of symmetrical dimer channels. Lipid vesicles containing gramicidin were dialyzed against control vesicles without gramicidin in the presence of a constant amount of radioactive ²⁰¹Tl⁺ and increasing amounts of non-radioactive Tl⁺. The ratio of ²⁰¹Tl⁺ free in solution to ²⁰¹Tl⁺ bound to the channel was measured after equilibrium (≥ 48 h) at 23 °C, and this ratio was plotted as a function of the free Tl⁺ concentration. The inverse of the slope yielded 0.8 to 1.1 for the maximum number of simultaneously occupied highest affinity sites per channel, and the inverse of the intercept yielded a highest affinity constant of 500 to 1000 m^?1 for each site. It appears that direct electrostatic repulsion prevents ions from binding simultaneously to the identical channel ends for thallium ion concentrations up to 20 mm. Estimates of the highest affinity constants for Rb⁺ and Na⁺ were also obtained.     

Determination of the molecular conformation of beta-D-O2,2'-cyclouridine in aqueous solution by proton magnetic resonance spectroscopy

The solution conformation of β-D -O²,2′-cyclouridine has been determined at 27 and 88°C in D₂O by proton magnetic resonance spectroscopy. The conformation is described in terms of a fixed syn-like sugar-base torsional angle, a type S furanose ring conformation (similar to 2′-endo), and a temperature-dependent exocyclic C(4)′–C(5′) rotamer population containing approximately 50% of the gauche-gauche form at 27°C. β-D -O²,2′-Cyclouridine 5′-phosphate likewise possesses a type S furanose ring conformation.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

The effect of gramicidin on ATP synthesis in pea chloroplasts: two modes of phosphorylation

The effect of gramicidin on the phosphorylation rate, electron flow and light-induced H²⁺ uptake (ΔH⁺) in chloroplasts with methyl viologen (MV) or phenazine methosulfate (PMS) added has been studied. In the presence of MV low concentrations of gramicidin (~10⁻⁹ M) were shown to inhibit the phosphorylation rate up to 80–90% of the initial value, without changing either the electron transport rate or ΔH⁺ value. Two components of phosphorylation have been identified in the presence of PMS - the first is DCMU-sensitive, which was suppressed by low gramicidin concentrations (< 10⁻⁹ M) and the second - DCMU-insensitive component, which was suppressed by high gramicidin concentrations (~10⁻⁷ M) exclusively, the high gramicidin concentration inducing the ΔH⁺ value fo fall.