Simultaneous fluorescence and conductance studies of planar bilayer membranes containing a highly active and fluorescent analog of gramicidin A

Veatch, William R.; Mathies, R. A.; Eisenberg, Moisés; Stryer, Lubert

doi:10.1016/s0022-2836(75)80160-4

Cited by 185 publications

(141 citation statements)

References 18 publications

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“…Simultaneous fluorescence and conductance studies of planar bilayer membranes containing the analogue dansyl-gramicidin C were reported by Veatch et al (30). On the basis of their results with 1,2-dioleoyl-glycero-phosphocholine (1,2-DOPC)/ decane membranes (cf.…”

mentioning

confidence: 87%

Lipid phase transition in planar bilayer membrane and its effect on carrier- and pore-mediated ion transport.

Boheim¹,

Hanke²,

Eibl³

1980

Proc. Natl. Acad. Sci. U.S.A.

127

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Using mixed-chain lipids, we have recorded cooling and heating curves of planar bilayer membranes while they passed the lipid phase transition range. With unmodified planar bilayers, spontaneous current fluctuations are observed near the lipid phase transition temperature (tc t 290C). This effect coincides with the expected and measured decrease in membrane capacitance. Carrier (valinomycin)-modified planar bilayers exhibit near tc an abrupt change from a high-conducting state above tc to the state ofbare membrane conductance below tc. In contrast to this behavior, planar bilayers modified by pore-forming antibiotics (gramicidin A, alamethicin) do not show any peculiar effect at tc. However, at 22-230C a pronounced maximum in pore-induced conductance is seen. Whereas the gramicidin A pore abruptly stops stepwise fluctuations below 16'C, with alamethicin a few long-lasting pore and pore state fluctuations persist down to 10'C. It is suggested that the carrier may freeze out into the membrane/water interface. The effects observed with pore-forming substances, on the other hand, are interpreted in terms of lateral phase separation into pure lipid and lipid/antibiotic domains. Phase changes are well-known phenomena in artificial lipid/ water systems (1) and biological systems (2). These phase transitions, which may play a role in triggering biological processes (3), can be induced by temperature changes or by interaction of ions with charged membrane lipids (4, 5). A great number of publications report on phase transition phenomena in pure and protein-loaded vesicles and liposomes. Due to the instability of planar bilayer membranes in the solid state, there are so far only two reports on electrochemical measurements in the freezing and melting range of planar lipid bilayer membranes. Experiments carried out on membranes from a 1:1 (wt/wt) mixture of dipalmitoylglycerol and distearoylglycerol in n-decane led to the interpretation that ion carriers became frozen and thus immobile within the membrane phase (6). On the other hand, the ionic conductance induced by the pore-former gramicidin was found to remain unchanged at the transition temperature tc of 41'C. Recently, ion-conducting channels were reported to appear in unmodified planar bilayer membranes at the phase tr of 59°C (7). Membranes were formed from a 1,2-distearoyl-glycero-3-phosphocholine/decane solution. There is also a paper, based on optical reflectivity measurements on membranes from monostearoylglycerol in n-hexadecane, which demonstrated an ;70% increase in membrane thickness when the system was cooled below the tc of 55°C (8).Using saturated mixed-chain lipids with a tc of ;290C, we succeeded in forming virtually solvent-free planar bilayer membranes below and above tc. In this paper we report our investigations on pure and ionophore-modified planar bilayers of this type in the 10-40°C temperature range.

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mentioning

confidence: 87%

Lipid phase transition in planar bilayer membrane and its effect on carrier- and pore-mediated ion transport.

Boheim¹,

Hanke²,

Eibl³

1980

Proc. Natl. Acad. Sci. U.S.A.

127

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“…Furthermore, combined conductance and spectroscopic measurements showed that the channels are dimers, and that all dimers are conducting channels 10 This allowed for nuclear magnetic resonance experiments, using 13 C and 19 F NMR to probe the exposure of the N-and C-termini to lipid or water-soluble shift reagents 11 , which showed conclusively that membranespanning gramicidin channels are head-tohead SS dimers. Later experiments show that circular dichroism spectra of bilayerincorporated gramicidin differ qualitatively from those of gramicidin in organic solvents 12 and that DS dimers are characteristic for gramicidin in organic solvents 13 .…”

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confidence: 96%

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et al. 1999

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“…Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across membranes (1)(2)(3)(4) by forming transmembrane channels (4,5), each of which is made up of two molecules of gramicidin (6)(7)(8)(9)(10) Veatch et al (7) demonstrated that, if all of the gramicidin in a planar bilayer membrane is dimerized, all of the gramicidin forms ion-conducting channels. Further, Veatch and Stryer (8) used fluorescence energy transfer to show that gramicidin is dimerized on phosphatidylcholine liposomes.…”

mentioning

confidence: 99%

Conformation of gramicidin A channel in phospholipid vesicles: a 13C and 19F nuclear magnetic resonance study.

Weinstein¹,

Wallace²,

Blout³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

145

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We have determined the conformation of the channel-forming polypeptide antibiotic gramicidin A in phosphatidylcholine vesicles by using '3C and 19F NMR spectroscopy. The models previously proposed for the conformation of the dimer channel differ in the surface localization of the NH2 and COOH termini. We have incorporated specific 13C and 19F nuclei at both the NH2, and COOH termini of gramicidin and have used 13C and 19F chemical shifts and spin lattice relaxation time measurements to determine the accessibility of these labels to three paramagnetic NMR probes-two in aqueous solution and one attached to a phosphatidylcholine fatty acid chain. All of our results indicate that the COOH terminus of gramicidin in the channel is located near the surface of the membrane and the NH2 terminus is buried deep within the lipid bilayer. These findings strongly favor an NH2-terminal to NHZ-terminal helical dimer as the major conformation for the gramicidin channel in phosphatidylcholine vesicles.Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across membranes (1-4) by forming transmembrane channels (4, 5), each of which is made up of two molecules of gramicidin (6-10). The amino acid sequence of valine gramicidin A (11) is:HCOVeatch et al. (7) The indole protons of gramicidin were resolved from the lipid resonances. It was argued that these indole protons were moderately exposed to the solution, on the basis of their Tm3+-induced chemical shift change; however, due to the lack of markers on or near the NH2 terminus, specific conformational conclusions could not be drawn. It should be noted that the nominal NH2 terminus is, in fact, formylated, and the nominal COOH terminus is, in fact, an ethanolamide.Four classes of models have been proposed for the conformation of the dimer gramicidin transmembrane channel ( (15) as one of the dimer conformations found for gramicidin in nonpolar organic solvents (15, 16). Model D is a parallel-f double helix with both NH2 termini at one end of the channel. A distinctive feature of both double-helical models is the presence of both the NH2 and COOH termini at the surfaces of the membrane (Fig. 1 C and D). For the NH2-terminal to NH2-terminal dimer (Fig. 1A) only the COOH termini are on the surfaces, and for the COOH-terminal to COOH-terminal dimer (Fig. 1B) only the NH2 termini are on the surface.We have incorporated specific 13C and 19F nuclei at both the NH2 and COOH termini (Fig. 2) and have carried out NMR experiments to determine the relative accessibility of these 13C and 19F nuclei to paramagnetic NMR probes. Two of these probes are localized in the aqueous solution-manganous ion (Mn2+) and thulium ion (Tm3+)-and the third is localized in the membrane interior-a nitroxide spin label covalently att Present address:

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Simultaneous fluorescence and conductance studies of planar bilayer membranes containing a highly active and fluorescent analog of gramicidin A

Cited by 185 publications

References 18 publications

Lipid phase transition in planar bilayer membrane and its effect on carrier- and pore-mediated ion transport.

Lipid phase transition in planar bilayer membrane and its effect on carrier- and pore-mediated ion transport.

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Conformation of gramicidin A channel in phospholipid vesicles: a 13C and 19F nuclear magnetic resonance study.

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