Nonstatistical UV Fragmentation of Gas-Phase Peptides Reveals Conformers and Their Structural Features.

Solving the 3D structure of a biomolecule requires recognition of its conformers and measurements of their individual structural identities, which can be compared with calculations. We employ the phenomenon of nonstatistical photofragmentation, detected by a combination of UV cold ion spectroscopy and high-resolution mass spectrometry, to identify the main conformers of gas-phase peptides and to recover individual UV absorption and mass spectra of all of these conformers in a single laser scan. We first validate this approach with a benchmark dipeptide, Tyr-Ala, and then apply it to a decapeptide, gramicidin S. The revealed characteristic structural difference between the conformers of the latter identifies some of the previously calculated structures of gramicidin S as the most likely geometries of its remaining unsolved conformer.

Mechanistic insight into gramicidin-based detection of protein-ligand interactions via sensitized photoinactivation.

Among the many challenges for the development of ion channel-based sensors is the poor understanding of how to engineer modified transmembrane pores with tailored functionality that can respond to external stimuli. Here, we use the method of sensitized photoinactivation of gramicidin A (gA) channels in planar bilayer lipid membranes to help elucidate the underlying mechanistic details for changes in macroscopic transmembrane ionic current observed upon interaction of C-terminally attached gA ligands with specific proteins in solution. Three different systems were studied: (i) carbonic anhydrase (CA) and gA-sulfonamide, (ii) PSD-95 protein (belonging to the 'PDZ domain-containing protein') and a gA analog carrying the KGGHRRSARYLESSV peptide sequence at the C-terminus, and (iii) an anti-biotin antibody and gA-biotin. The results challenge a previously proposed mechanistic hypothesis suggesting that protein-induced current suppression is due to steric blockage of the ion passage through gA channels, while they reveal new insight for consideration in alternative mechanistic models. Additionally, we demonstrate that the length of a linker between the ligand and the gA channel may be less important for gramicidin-based detection of monovalent compared to multivalent protein-ligand interactions. These studies collectively shed new light on the mechanism of protein-induced current alterations in bilayer recordings of gA derivatives, which may be important in the design of new gramicidin-based sensors.

Specific photodissociation of peptides with multi-stage mass spectrometry.

We report collision-induced dissociation (CID) and laser-induced dissociation (LID) performed at different wavelengths between 220 and 280 nm of the peptides leucine-enkephalin (protonated) and gramicidin A (sodiated). Hydrogen-atom losses and side-chain cleavages were observed in LID experiments. These losses depend on the laser wavelength and lead to the formation of radical ions. The fragmentations of these radicals, which are not observed in CID experiments, were investigated in multi-stage mass spectrometry experiments.

Kinetically different populations of O-pyromellityl-gramicidin channels induced by poly-L-lysines in lipid bilayers.

Clustering of membrane proteins, in particular of ion channels, plays an important role in their functioning. To further elucidate the mechanism of such ion channel activity regulation, we performed experiments with a model system comprising the negatively-charged gramicidin analog, O-pyromellitylgramicidin (OPg) that forms ion channels in bilayer lipid membrane (BLM), and polycations. The effect of polylysines on the kinetics of OPg channels in BLM was studied by the method of sensitized photoinactivation. As found in our previous work, the interaction of polylysine with OPg led to the deceleration of the OPg photoinactivation kinetics, i.e., to the increase in the characteristic time of OPg photoinactivation. It was shown here that in a certain range of polylysine concentrations the photoinactivation kinetics displayed systematic deviations from a monoexponential curve and was well described by a sum of two exponentials. The deviations from the monoexponential approximation were more pronounced with polylysines having a lower degree of polymerization. These deviations increased also upon the elevation of the ionic strength of the bathing solution and the addition of calcium ions. A theoretical model is presented that relates the OPg photoinactivation kinetics at different concentration ratios of OPg and polylysine to the distribution of OPg molecules among OPg-polylysine clusters of different stoichiometry. This model is shown to explain qualitatively the experimental results, although the quantitative description of the whole body of evidence requires further development, assuming that the interaction of polylysine with OPg causes segregation of membrane domains enriched in OPg channels. The single-channel data, which revealed the insensitivity of the single-channel lifetime of OPg to the addition of polylysine, are in good agreement with the theoretical model.

Effect of fluoride anions on gramicidin photoinactivation sensitized by sulfonated aluminum phthalocyanines.

Interaction of potent photodynamic agents, sulfonated aluminum phthalocyanines (AlPcSn where n is a number of sulfonic groups), with biological membranes was studied here using model systems: sensitized photoinactivation of gramicidin channels in planar lipid bilayers and adsorption on lipid monolayers. Fluoride anions known to form complexes with aluminum were found to inhibit both the adsorption of aluminum phthalocyanines on lipid monolayers, as measured with a Langmuir trough by surface pressure and surface potential changes, and photodynamic efficacy of the dyes, as studied by gramicidin channel photoinactivation. The similar effects were caused by the alkalinization of the medium. Fluoride anions appeared to be much more effective in the case of AlPcS4 as compared to AlPcS3. The suppression of the photodynamic potency of aluminum phthalocyanines was attributed to desorption of the dyes from lipid bilayers induced by fluoride or hydroxyl ions. With AlPcS4 an enhancement of the dye aggregation leading to a decrease in the sensitizing activity was probably involved in the fluoride effect as revealed by absorption and fluorescence spectral measurements. Capillary electrophoresis was employed to understand the mechanism of the dye desorption. The results of these experiments indicated that the reduction in the membrane affinity was associated with an increase in the negative charge of the dye molecules due to the binding of fluoride or hydroxyl ions.

Photodynamic and radiolytic inactivation of ion channels formed by gramicidin A: oxidation and fragmentation.

Ion channels formed by the peptide gramicidin A in planar lipid membranes have been reported to react very sensitively upon irradiation of the membrane by ionizing radiation (radiolysis), by UV light (photolysis), or by visible light in the presence of appropriate photosensitizers (photodynamic inactivation). In all three cases the effect is due to the presence of the four tryptophan residues of the pentadecapeptide. Modifications of these amino acids--due to an interaction with free radicals formed upon water radiolysis or due to light absorption--have been found to reduce the membrane conductance by many orders of magnitude. The present study was intended to correlate functional changes, observed at the level of single ion channels, with changes of the molecular structure identified by mass spectrometry. About 98% of the inactivated channels showed a single-channel conductance of virtually zero, while about 2% of the channels present before irradiation are converted to a state of reduced conductance (and reduced lifetime). On the structural level, irradiation in the presence of the photosensitizer Rose Bengal was found to produce oxidation and fragmentation of the peptide at the positions of the tryptophan residues. Our results provide evidence that the main effect of radiolysis, or of photodynamic treatment, is the cleavage of the peptide backbone leading to immediate closure of an open ion channel.

Absence of effects of low-frequency, low-amplitude magnetic fields on the properties of gramicidin A channels.

The effects of static and low-frequency magnetic fields on gramicidin A channels have been investigated using bilayer patch clamp recording and a bridge technique capable of detecting 0.3% changes in the conductance of glyceryl monooleate membranes containing many channels. In the bridge technique the conductance was assessed using 10-ms voltage pulses applied at 10 Hz. Measurements were made for LiCl, KCl, and CsCl using magnetic fields of 50, 100, 500, and 5000 microT with the frequency scanned from 10-200 Hz. The combinations of static and low-frequency fields employed include the "cyclotron resonance" conditions at which effects had been predicted to occur. In no case was there any detectable change in conductance when the magnetic fields were applied or changed. Potassium currents through single gramicidin channels have been recorded for patches in which several channels may be open at once. Fields were applied for 2 min periods interleaved with 2 min controls. Methods have been developed to analyze the multichannel records to reveal the amplitude and duration of the channels together with the frequency, depth, and apparent period of flickers. No significant differences were observed between the control and field-exposed recording periods. The peak of the distribution of opening and closing transitions always coincided for fields on and off within the resolution, 0.4%, of the recordings. There are at least two types of flicker, one with typical period less than 0.1 ms, the other with typical period from 0.3-0.8 ms. Most of the latter were not complete closures with the conductance during a flicker 15-20% above the level for a full closure.

Direct observation of differential UV photolytic degradation among the tryptophan residues of gramicidin A in sodium dodecyl sulfate micelles.

Gramicidin A, incorporated into sodium dodecyl sulfate micelles, was exposed to ultraviolet light and discovered by two-dimensional (TOCSY) NMR spectroscopy to undergo differential photolytic degradation. The four tryptophan residues of gramicidin A were found to be unequally sensitive to ultraviolet radiation. Tryptophan 9 was the most sensitive to ultraviolet photolysis, while tryptophan 11 was the least sensitive. Tryptophans 13 and 15 have approximately the same susceptibility to photolytic degradation by the ultraviolet light. Rate constants for the photolytic degradation of the four tryptophan residues were obtained from the dependence of the TOCSY spectrum upon the time of photolysis.

Photodynamic inactivation of an ion channel: gramicidin A.

The ion channel formed by the peptide gramicidin A in planar lipid membranes is inactivated by visible light in the presence of the photosensitizer Rose Bengal. This is concluded from the strong decrease of the membrane conductance by more than two orders of magnitude. Experiments performed at different oxygen concentrations, in the presence of the singlet oxygen quenchers beta-carotene or alpha-tocopherol indicate, that presumably a type I process between the dye Rose Bengal and the tryptophan residues of the gramicidin channel with a subsequent oxidation of the tryptophans is responsible for the loss of the conductance properties of the channel.

[The radiobiological characteristics of the P+ variant of Bacillus brevis var. G.--B. in relation to gramicidin S synthesis]. / Radiobiologicheskie osobennosti P+-varianta Bacillus brevis var. G.--B.v sviazi s sintezom gramitsidina S.

The radiosensitivity of P(+) variant Bacillus brevis var. G.-B. cells cultured under condition of normal and inhibited gramicidin S synthesis, antibiotically high-active strain and high radioresistant cells has been studied. It has been shown that the radioresistance of bacterial cells correlates, in general, with their antibiotic activity: the antibiotic superproduced is more radioresistant than P(+) variant, the inhibition of antibiotic synthesis by beta-phenil-beta-alanin rises a little the sensitivity of P(+) variant cells. But the radioresistant fraction of P(+) variant contains the lower antibiotic amount than the whole population. It has been concluded that the radioprotective action of gramicidin S can not be the only reason of the above-mentioned differences in radiosensitivity.

Effects of ionizing radiation on artificial (planar) lipid membranes. I. Radiation inactivation of the ion channel gramicidin A.

The ion channel formed by the pentadecapeptide gramicidin A in planar lipid membranes is extremely sensitive to ionizing radiation. The membrane conductance may drop by several orders of magnitude under appropriate experimental conditions (low pH and presence of oxygen). The radiation sensitivity is strongly reduced for gramicidin M-. This analogue has the four tryptophan residues replaced by phenylalanines. Experiments performed in the presence of various radical scavengers suggest that the inactivation of the channel is due to a combined action of OH and of HO2 radicals at the tryptophan residues. The shape of the inactivation curves following continuous radiolysis or pulse radiolysis were found to be in fair agreement with a simple model which assumes that the damage of a single tryptophan residue is sufficient for channel inactivation. The conductance of inactivated channels could not be resolved within the experimental accuracy. This is contrary to photolysis of gramicidin channels found by Busath and Waldbilling (1983), where a broad distribution of low conductance states was observed. The inactivation by radiolysis seems to represent an 'all-or-none-process' of the channel conductance.