Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers (original) (raw)
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Monitoring Gramicidin Conformations in Membranes: A Fluorescence Approach
Biophysical Journal, 2004
We have monitored the membrane-bound channel and nonchannel conformations of gramicidin utilizing rededge excitation shift (REES), and related fluorescence parameters. In particular, we have used fluorescence lifetime, polarization, quenching, chemical modification, and membrane penetration depth analysis in addition to REES measurements to distinguish these two conformations. Our results show that REES of gramicidin tryptophans can be effectively used to distinguish conformations of membrane-bound gramicidin. The interfacially localized tryptophans in the channel conformation display REES of 7 nm whereas the tryptophans in the nonchannel conformation exhibit REES of 2 nm which highlights the difference in their average environments in terms of localization in the membrane. This is supported by tryptophan penetration depth measurements using the parallax method and fluorescence lifetime and polarization measurements. Further differences in the average tryptophan microenvironments in the two conformations are brought out by fluorescence quenching experiments using acrylamide and chemical modification of the tryptophans by N-bromosuccinimide. In summary, we report novel fluorescence-based approaches to monitor conformations of this important ion channel peptide. Our results offer vital information on the organization and dynamics of the functionally important tryptophan residues in gramicidin.
Gramicidin channels in phospholipid bilayers with unsaturated acyl chains
Biophysical Journal, 1997
In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded 36-3-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded 7rr5r6-helical dimers predominate (over single-stranded f6_3-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1 ,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded 7r7r5_6-helical dimers in any of these different membrane environments. We conclude that the single-stranded p663-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.
Effect of acyl chain length on the structure and motion of gramicidin A in lipid bilayers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1989
The transmembrane ion transport properties of gramicidin A have previously been shown to be dependent on the nature of its lipid environment. Solid-state NMR spectroscopic studies of I'~C-labelled znalogues of gramicidin in oriented multilayers of phosphatidyleholine have shown that variation of the lipid hydrocarbon chain length has no effect on the structure or orientation of the peptide backbone.
Channel and Nonchannel Forms of Spin-Labeled Gramicidin in Membranes and Their Equilibria
The Journal of Physical Chemistry B, 2011
Channel and non-channel forms of gramicidin A (GA) were studied by ESR in various lipid environments using new mono-and double-spin labeled compounds. For GA channels we demonstrate here how Pulse Dipolar ESR can be used to determine the orientation of the membrane-traversing molecule relative to the membrane normal and to study subtle effects of lipid environment on the interspin distance in the spin labeled gramicidin channel. To study non channel forms of gramicidin, Pulse Dipolar ESR was used first to determine interspin distances corresponding to monomers and double helical dimers of spin labeled GA molecules in the organic solvents trifluoroethanol and octanol. The same distances were then observed in membranes. Since detection of non-channel forms in the membrane is complicated by aggregation, we suppressed any dipolar spectra from intermolecular interspin distances arising from the aggregates by using double-labeled GA in a mixture with excess unlabeled GA. In hydrophobic mismatching lipids (L β phase of DPPC) gramicidin channels dissociate into free monomers. The backbone structure of the monomeric form is similar to a monomeric unit of the channel dimer. In addition to channels and monomers the double helical conformation of gramicidin is present in some membrane environments. In the gel phase of saturated phosphatidylcholines the fraction of double helices increases in the following order: DLPC<DMPC<DSPC<DPPC. The equilibrium DHD/monomer ratio in DPPC was determined. In membranes the double helical form is present only in aggregates. In addition, we studied the effect of N-terminal substitution in the GA molecule upon channel formation. This work demonstrates how Pulsed Dipolar ESR may be utilized to study complex equilibria of peptides in membranes.
International Journal of Molecular Sciences, 2018
When using small mole fraction increments to study gramicidins in phospholipid membranes, we found that the phasor dots of intrinsic fluorescence of gramicidin D and gramicidin A in dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar and multilamellar vesicles exhibit a biphasic change with peptide content at 0.143 gramicidin mole fraction. To understand this phenomenon, we developed a statistical mechanical model of gramicidin/DMPC mixtures. Our model assumes a sludge-like mixture of fluid phase and aggregates of rigid clusters. In the fluid phase, gramicidin monomers are randomly distributed. A rigid cluster is formed by a gramicidin dimer and DMPC molecules that are condensed to the dimer, following particular stoichiometries (critical gramicidin mole fractions, Xcr including 0.143). Rigid clusters form aggregates in which gramicidin dimers are regularly distributed, in some cases, even to superlattices. At Xcr, the size of cluster aggregates and regular distributions reac...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
The mode of orientation of gramicidin A in the •6.3 conformation in a monolayer of lysophosphatidylcholine was studied on the basis of conformational data. The approach involves localization of the centers of hydrophobicity and hydrophilicity of the gramicidin molecule, and takes into account the dielectric constant discontinuity of the interface. The most probable orientation is that with the C-terminal, tryptophan-rich, end directed towards the hydrophobic medium. In this orientation, four lysophosphatidylcholines surround a gramicidin monomer leading to a complex with an overall cylindrical shape compatible with the bilayer structure found in aqueous dispersions of mixtures of these molecules (
Biophysical Journal, 1999
We present a quantitative analysis of the effects of hydrophobic matching and membrane-mediated proteinprotein interactions exhibited by gramicidin embedded in dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) bilayers (Harroun et al., 1999. Biophys. J. 76:937-945). Incorporating gramicidin, at 1:10 peptide/lipid molar ratio, decreases the phosphate-to-phosphate (PtP) peak separation in the DMPC bilayer from 35.3 Å without gramicidin to 32.7 Å. In contrast, the same molar ratio of gramicidin in DLPC increases the PtP from 30.8 Å to 32.1 Å. Concurrently, x-ray in-plane scattering showed that the most probable nearest-neighbor separation between gramicidin channels was 26.8 Å in DLPC, but reduced to 23.3 Å in DMPC. In this paper we review the idea of hydrophobic matching in which the lipid bilayer deforms to match the hydrophobic surface of the embedded proteins. We use a simple elasticity theory, including thickness compression, tension, and splay terms to describe the membrane deformation. The energy of membrane deformation is compared with the energy cost of hydrophobic mismatch. We discuss the boundary conditions between a gramicidin channel and the lipid bilayer. We used a numerical method to solve the problem of membrane deformation profile in the presence of a high density of gramicidin channels and ran computer simulations of 81 gramicidin channels to find the equilibrium distributions of the channels in the plane of the bilayer. The simulations contain four parameters: bilayer thickness compressibility 1/B, bilayer bending rigidity K c , the channel-bilayer mismatch D o , and the slope of the interface at the lipid-protein boundary s. B, K c , and D o were experimentally measured; the only free parameter is s. The value of s is determined by the requirement that the theory produces the experimental values of bilayer thinning by gramicidin and the shift in the peak position of the in-plane scattering due to membrane-mediated channel-channel interactions. We show that both hydrophobic matching and membrane-mediated interactions can be understood by the simple elasticity theory.
Biophysical Journal, 2004
Although there have been several decades of literature illustrating the opening and closing of the monovalent cation selective gramicidin A channel through single channel conductance, the closed conformation has remained poorly characterized. In sharp contrast, the open-state dimer is one of the highest resolution structures yet characterized in a lipid environment. To shift the open/closed equilibrium dramatically toward the closed
Biochemistry, 2011
We investigated the effects of substituting two of the four tryptophans (the "inner pair" Trp 9,11 or the "outer pair" Trp 13,15 ) in gramicidin A (gA) channels. The conformational preferences of the double-substituted gA analogues were assessed using circular dichroism spectroscopy and sizeexclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA's conformational preference in lipid bilayer membranes. [Phe 13, ]gA largely retains the single-stranded helical channel structure, whereas of [Phe 9,11 ]gA exists primarily as doublestranded conformers. Within this context, the 2 H-NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state 2 H-NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phesubstituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe 13,15 ]gA channels have Na + currents that arẽ 50% and lifetimes ~80% those of native gA channels. The double-stranded conformer(s) of [Phe 9,11 ]gA do not form detectable channels. The minor single-stranded population of [Phe 9,11 ]gA forms channels with Na + currents that are ~25% and single-channel lifetimes that are ~300% those of native gA channels. Our results suggest that Trp 9 and Trp 11 , when "reaching" for the interface, tend to drive both monomer folding (to "open" a channel) and dimer dissociation (to "close" a channel). Furthermore, the dipoles of Trp 9 and Trp 11 are relatively more important for the singlechannel conductance than are the dipoles of Trp 13 and Trp 15 .