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R E Koeppe

Publications and source records attributed to R E Koeppe.

At least 19 recordsLinked to original sources
PubMed1992

Orientation of the valine-1 side chain of the gramicidin transmembrane channel and implications for channel functioning. A 2H NMR study.

The orientation of the valine-1 side chain of gramicidin was determined by solid-state 2H NMR using valine-1-deuterated (d8) gramicidin. The peptide was incorporated into DMPC bilayers that were oriented between glass plates. When the plates were oriented with their normal perpendicular to the magnetic field, four quadrupolar splittings were observed of 106, 68, 9.7, and 2.0 kHz. These resonances were assigned to C alpha D, C beta D, and the deuterons of each of the C gamma D3 methyl groups, respectively. The average orientation of the various C-D bonds was calculated with respect to the helix axis. The angle obtained for the C alpha-D resonance was consistent with a single-stranded beta 6.3-helical model for the backbone but not with double-helical models. The angles of the side chain were then fitted to a model for the right-handed beta 6.3-helix. Rotation of the valine-1 side chain yielded a set of torsion angles that matched the angles as determined from the 2H NMR measurements. The corresponding orientation of the valine-1 side chain (chi 1 = -5 degrees) was found to be quite unusual, but it explains well the importance of a branched side chain at position 1 for channel formation and stability. A van der Waals interaction between valine-1 of one monomer and alanine-5 of the other helps to stabilize the gramicidin dimer.

Amino Acid Sequence↗
PubMed1992

Molecular and channel-forming characteristics of gramicidin K's: a family of naturally occurring acylated gramicidins.

The gramicidin K family is a set of naturally occurring acylated linear peptides in which a fatty acid is esterified to the ethanolamine hydroxyl of either gramicidin A or C, and possibly also to gramicidin B (Koeppe, R. E., II, Paczkowski, J. A., & Whaley, W. L. (1985) Biochemistry 24, 2822-2826). These acylated gramicidins form membrane-spanning channels in planar lipid bilayers and therefore constitute a model system with which to study the structural and functional consequences of acylation on membrane proteins. This paper serves to characterize further the channels formed by acylated gramicidins A and C and to demonstrate that these channels are structurally equivalent to the channels formed by the standard gramicidins. We also present additional evidence for the ester linkage in the natural acylated gramicidins A and C and identify the fatty acyl chains.

Acylation↗
PubMed1992

On the helix sense of gramicidin A single channels.

In order to resolve whether gramicidin A channels are formed by right- or left-handed beta-helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therefore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A- and M- channels are structurally equivalent, while gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural gramicidin A channels in phospholipid bilayers are right-handed beta 6.3-helical dimers.

Amino Acid Sequence↗
PubMed1992

Formation of non-beta 6.3-helical gramicidin channels between sequence-substituted gramicidin analogues.

Using the linear gramicidins as an example, we have previously shown how the statistical properties of heterodimeric (hybrid) channels (formed between the parent [Val1]gramicidin A (gA) and a sequence-altered analogue) can be used to assess whether the analogue forms channels that are structurally equivalent to the parent channels (Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. J. Mol. Biol. 211:221-234). Generally, the gramicidins are tolerant of amino acid sequence alterations. We report here an exception. The optically reversed analogue, gramicidin M- (gM-) (Heitz, F., G. Spach, and Y. Trudelle. 1982. Biophys. J. 40:87-89), forms channels that are the mirror-image of [Val1]gA channels; gM- should thus form no hybrid channels with analogues having the same helix sense as [Val1]gA. Surprisingly, however, gM- forms hybrid channels with the shortened analogues des-Val1-[Ala2]gA and des-Val1-gC, but these channels differ fundamentally from the parent channels: (a) the appearance rate of these heterodimers is only approximately 1/10 of that predicted from the random assortment of monomers into conducting dimers, indicating the existence of an energy barrier to their formation (e.g., monomer refolding into a new channel-forming conformation); and (b), once formed, the hybrid channels are stabilized approximately 1,000-fold relative to the parent channels. The increased stability suggests a structure that is joined by many hydrogen bonds, such as one of the double-stranded helical dimers shown to be adopted by gramicidins in organic solvents (Veatch, W. R., E. T. Fossel, and E. R. Blout. 1974. Biochemistry. 13:5249-5256).

Amino Acid Sequence↗
PubMed1992

On the supramolecular organization of gramicidin channels. The elementary conducting unit is a dimer.

The question, whether the conducting channels formed by the linear gramicidins are dimers (as is generally believed) or tetramers (as has been recently proposed [Stark G., M. Strässle, and Z. Takacz. 1986. J. Membr. Biol. 89:23-37; Strässle, M., G. Stark, M. Wilhelm, P. Daumas, F. Heitz, and R. Lazaro. 1989. Biochim. Biophys. Acta. 980:305-314]) has been addressed in single-channel experiments. The experimental approach was based on the ability of electrophysiological (single-channel) experiments to resolve the number of hybrid channel types that could form between gramicidin A or C and O-pyromellityl-gramicidin A or C (in which a pyromellitic acid residue has been esterified to the ethanolamine-OH group [Apell, H.-J., E. Bamberg, H. Alpes, and P. Läuger. 1977. J. Membr. Biol. 31:171-188]). The presence of the bulky, negatively charged pyromellityl group at the channel entrances endows the hybrid channels with characteristically different features and thus facilitates the resolution of the different hybrid channel types. Only two hybrid channel types were detected, indicating that the conducting channels are membrane-spanning dimers. There was likewise no evidence for lateral association between conducting channels and nonconducting monomers. These results can be reconciled with those of Stark et al. (op. cit.) if gramicidin channel formation involves a (slow) folding into beta 6.3-helical monomers followed by the dimerization step.

Gramicidin↗
PubMed1992

Molecular determinants of channel function.

The 40 years since the seminal papers of Hodgkin and Huxley appeared have been extraordinarily productive in terms of understanding the molecular basis for electrical activity. The Hodgkin-Huxley proposal that electrical excitability should be understood in terms of voltage-dependent changes in discrete sites has been resoundingly verified. Indeed, the Hodgkin-Huxley framework is remarkable in that its essential elements have remained largely intact as molecular understanding has advanced. This robustness is, at least in part, a result of the fact that Hodgkin and Huxley developed a mathematical model, based on simple physical arguments, that was sufficiently comprehensive to describe the kinetics of the voltage-clamped currents and yet simple enough to be predictive. The predictive features were demonstrated early by the reconstruction of both space-clamped and propagated action potentials on a desk-top calculator (293) and, later, when the sites of Hodgkin and Huxley developed into being well-characterized molecular structures. Voltage- and ligand-dependent ion-selective channels are now the established framework within which cellular electrophysiology is being pursued. Moreover, electrophysiological measurements of membrane and single-channel currents have become essential tools to examine molecular questions pertaining to channel structure and activity. The last 10 years have witnessed spectacular activity, which has resulted from two developments, the giga-seal patch clamp (249) and the elucidation of primary sequences of a number of channel-forming proteins (494), along with the first outlines of their low-resolution three-dimensional structures (651). The stage is now set for 1) applying a variety of convergent techniques to decipher molecular structural details at high resolution, and 2) seeking to understand the complex dynamic functions, gating, and ion selectivity at the molecular level. The early successes are likely to be in understanding the molecular determinants of ion conductance and selectivity, initially in terms of quantitative descriptions of how a sequence modification can alter a channel's permeability characteristics. Channel gating is a far more elusive target because it involves molecular rearrangements, which are poorly understood at any level of description and which may be modified by the channel's environment. The general mechanisms of ion permeation and gating will differ among different classes of ion channels, but a molecular understanding of either phenomenon must eventually be based on an understanding of intermolecular forces, which are invariant among all channel types.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Amino acid sequence modulation of gramicidin channel function: effects of tryptophan-to-phenylalanine substitutions on the single-channel conductance and duration.

Linear gramicidins with one, two, or three Trp----Phe substitutions in the gramicidin A sequence form beta 6.3-helical channels that have widely varying conductances and average durations. The variations in single-channel conductance and average duration are uncoupled. The single-channel conductance decreases as a monotonic function of the number of Trp----Phe substitutions, and the relative conductance decrease induced by a given Trp----Phe substitution is only weakly affected by substitutions at other positions. These results suggest that each Trp influences the conductance independently, most likely through electrostatic interactions between the Trp dipole(s) and the permeant ion (as was deduced previously for aromatic side-chain substitutions at position one [Koeppe, R. E., Mazet, J.-L., & Andersen, O. S. (1990) Biochemistry 29 (2), 512-520]). Trp----Phe substitutions exert a complex, nonadditive influence on average duration as well as the energetics of heterodimer formation. These changes are presumably due to sequence-specific differences in the channel's surface chemistry--which may be related to ability of the Trp indole NH moieties to form hydrogen bonds with the lipid backbone oxygens and/or interfacial H2O.

Amino Acid Sequence↗
PubMed1991

Effect of salt and membrane fluidity on fluorophore motions of a gramicidin C derivative.

We have used fluorescence spectroscopy to investigate the effect of salt and membrane fluidity on the rotational motion of a 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) derivative of gramicidin C (dansyl-gC) in dimyristoylphosphatidylcholine bilayers, under conditions where the peptide is a formyl-NH to formyl-NH terminal dimer, and in octyl glucoside micelles, where the peptide is an intertwined helical dimer. Energy-transfer experiments showed no changes in either conformation or dimer aggregation under the conditions explored here (15-40 degrees C, 1-350 bar, 0-0.33 M Mg2+, and 0-1 M Na+). The addition of permeable (Na+) or nonpermeable (Mg2+) ions did not affect the temperature or pressure behavior of dansyl rotation. However, fluorescence lifetime measurements indicated an increase in solvent accessibility in the presence of sodium. In bilayers, the temperature dependence of the fluorescence polarization and lifetime shows strong interactions between the dansyl residue and the peptide, and at no time did the dansyl motions become solvent controlled as has been observed for aqueous solvent peptides [Scarlata, S. F., Rholam, M., & Weber, G. (1984) Biochemistry 23, 6789]. In micelles, the change in rotational motion with temperature followed solvent expansion, showing that in this case the dansyl residue does not associate extensively with the peptide. Our results indicate that because of the extensive coupling between the dansyl residue and the rest of the peptide, membrane fluidity does not play a major role in controlling side-chain motions.

Amino Acid Sequence↗
PubMed1990

Kinetics of gramicidin channel formation in lipid bilayers: transmembrane monomer association.

Conducting gramicidin channels form predominantly by the transmembrane association of monomers, one from each side of a lipid bilayer. In single-channel experiments in planar bilayers the two gramicidin analogs, [Val1]gramicidin A (gA) and [4,4,4-F3-Val1]gramicidin A (F3gA), form dimeric channels that are structurally equivalent and have characteristically different conductances. When these gramicidins were added asymmetrically, one to each side of a preformed bilayer, the predominant channel type was the hybrid channel, formed between two chemically dissimilar monomers. These channels formed by the association of monomers residing in each half of the membrane. These results also indicate that the hydrophobic gramicidins are surprisingly membrane impermeant, a conclusion that was confirmed in experiments in which gA was added asymmetrically and symmetrically to preformed bilayers.

Amino Acid Sequence↗
PubMed1990

Distinction between dipolar and inductive effects in modulating the conductance of gramicidin channels.

The ion permeability of transmembrane channels formed by the linear gramicidins is altered by amino acid sequence substitutions. We have previously shown that the polarity of the side chain at position one is important in modulating a channel's conductance and ion selectivity [Russel et al. (1986) Biophys. J. 49, 673-686]. Changes in polarity could alter ion permeability by (through-space) ion-dipole interactions or by (through-bond) inductive electron shifts. We have addressed this question by investigating the permeability characteristics of channels formed by gramicidins where the NH2-terminal amino acid is either phenylalanine or one of a series of substituted phenylalanines: p-hydroxy-, p-methoxy-, o-fluoro-, m-fluoro-, or p-fluorophenylalanine. The electron-donating or -withdrawing nature, as quantified by the Hammett constant, ranges from -0.37 to +0.34 for these side chains. Channels formed by these gramicidins show a more than 2.5-fold variation in their Na+ conductance, but the conductance variations do not rank in the order of the Hammett constants of the side chains. Inductive effects cannot therefore be of primary importance in the modulation of the gramicidin single-channel conductance by these side chains. The results support previous suggestions that electrostatic interactions between side chain dipoles and permeating ions can modify the energy profile for ion movement through the gramicidin channel and thus alter the conductance.

Amino Acid Sequence↗
PubMed1990

Energetics of gramicidin hybrid channel formation as a test for structural equivalence. Side-chain substitutions in the native sequence.

To determine whether amino acid side-chain substitutions in linear gramicidins after the structure of membrane-spanning channels formed by the modified peptides, we have developed a quantitative measure of structural equivalence of the peptide backbone among gramicidin channels based on functional (single-channel) measurements. The experiments exploit the fact that gramicidin channels are symmetrical dimers, and that channels formed by different gramicidin analogues can be distinguished on the basis of their single-channel current amplitudes or durations. It is thereby possible to determine whether hybrid channels can form between chemically dissimilar peptides, i.e. whether the peptides can adapt to each other. Further, since the relative rates of channel formation as well as the relative concentrations of pure and hybrid channel types can be measured in the same membrane, these experiments provide a quantitative measure of the energetic cost of hybrid channel formation relative to the formation of the pure channels. For a wide variety of different side-chains, we find that substitutions as extreme as glycine to phenylalanine at position 1, at the join between the two monomers in a membrane-spanning dimer, incur no energetic cost for channel formation, which implies that channels formed by each of the modified peptides are structurally equivalent. In addition, the average durations of the hybrid channels (except those having tyrosine or hexafluorovaline at position 1) are intermediate to the average durations of the respective pure channel types, thus providing further evidence for structural equivalence among channels formed by sequence-substituted gramicidins.

Amino Acid Sequence↗
PubMed1990

Gramicidins A, B, and C form structurally equivalent ion channels.

The membrane structure of the naturally occurring gramicidins A, B, and C was investigated using circular dichroism (CD) spectroscopy and single-channel recording techniques. All three gramicidins form channels with fairly similar properties (Bamberg, E., K. Noda, E. Gross, and P. Läuger. 1976. Biochim. Biophys. Acta. 419:223-228.). When incorporated into lysophosphatidylcholine micelles, however, the CD spectrum of gramicidin B is different from that of gramicidin A or C (cf. Prasad, K. U., T. L. Trapane, D. Busath, G. Szabo, and D. W. Urry. 1983. Int. J. Pept. Protein Res. 22:341-347.). The structural identity of the channels formed by gramicidin B has, therefore, been uncertain. We find that when gramicidins A and B are incorporated into dipalmitoylphosphatidylcholine vesicles, their CD spectra are fairly similar, suggesting that the two channel structures could be similar. In planar bilayers, gramicidins A, B, and C all form hybrid channels with each other. The properties of the hybrid channels are intermediate to those of the symmetric channels, and the appearance rates of the hybrid channels (relative to the symmetric channels) corresponds to what would be predicted if all three gramicidin molecules were to form structurally equivalent channels. These results allow us to interpret the different behavior of channels formed by the three gramicidins solely on the basis of the amino acid substitution at position 11.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Gramicidin single-channel properties show no solvent-history dependence.

The structure of membrane-associated gramicidins can depend on the solvent in which they were dissolved prior to membrane incorporation (LoGrasso, P. V., F. Moll, and T. A. Cross 1988. Biophys. J. 54:259-267; Killian, J. A., K. U. Prasad, D. Hains, and D. W. Urry. 1988. Biochemistry. 27:4848-4855). The peptide's solvent history might thus affect the functional characteristics of gramicidin channels (op. cit.). We tested this proposal by examining the properties (conductance, conductance dispersity, and average duration) of channels formed by [Val1]gramicidin A that had been dissolved in eight different solvents. The peptide was incorporated into lipid bilayers either by addition to the aqueous phase (and subsequent adsorption to the membrane) or by cosolubilization with the membrane-forming phospholipid. When the peptide was cosolubilized with the phospholipid, the channel properties did not vary with the solvent used. When the peptide was dissolved in chloroform, benzene, or trifluoroethanol and added through the aqueous phase, the channel properties differed from those found when gramidicin was dissolved in methanol, ethanol, dioxane, dimethylsulfoxide, or ethylacetate. The changes observed with the former three solvents were reproduced by adding them to the aqueous phase, and are therefore due to the ability of these solvents to partition into the membrane and alter the channels' behavior.

Gramicidin↗
PubMed1989

Induction of conductance heterogeneity in gramicidin channels.

In previous work from our laboratory, 5-10% of the channels formed by [Val1]gramicidin A have conductances that fall outside the narrow range that conventionally has defined the standard gramicidin channel [e.g., see Russell et al. (1986) Biophys. J. 49, 673]. Reports from other laboratories, however, show that up to 50% of [Val1]gramicidin channels have conductances that fall outside the range for standard channels [e.g., see Prasad et al. (1986) Biochemistry 25, 456]. This laboratory-to-laboratory variation in the distribution of gramicidin single-channel conductances suggests that the conductance variants are induced by some environmental factor(s) [Busath et al. (1987) Biophys. J. 51, 79]. In order to test whether extrinsic agents can induce such conductance heterogeneity, we examined the effects of nonionic or zwitterionic detergents upon gramicidin channel behavior. In phospholipid bilayers, detergent addition induces many changes in gramicidin channel behavior: all detergents tested increase the channel appearance rate and average duration; most detergents decrease the conductance of the standard channel; and all but one of the detergents increase the conductance heterogeneity. These results show that the conductance heterogeneity can result from environmental perturbations, thus providing a possible explanation for the laboratory-to-laboratory variation in the heterogeneity of gramicidin channels. In addition, the differential detergent effects suggest possible mechanisms by which detergents can induce the conformational perturbations that result in gramicidin single-channel conductance variations.

Detergents↗