Biomedical subjects

R E Koeppe

Publications and source records attributed to R E Koeppe.

At least 19 recordsLinked to original sources

PubMed1996

Conformation of the acylation site of palmitoylgramicidin in lipid bilayers of dimyristoylphosphatidylcholine.

Gramicidin A(gA) can be palmitoylated by means of an ester linkage to the OH group of the terminal ethanolamine that sits at the membrane-water interface in the functional gA channel. We have investigated palmitoyl-gA as a model transmembrane acylprotein. Ethanolamine-d(4) (NH(2)CD(2)CD(2)OH) was incorporated into gA by total synthesis, and a portion of the labeled gA was palmitoylated. Solid-state (2)H-NMR spectra of acyl- and nonacyl-gA in hydrated dimyristoylphosphatidylcholine (DMPC) bilayers were compared. The spectra for both oriented and nonoriented samples at 4 and at 40 degrees C indicate that the ethanolamine of gA is highly mobile prior to acylation, but essentially immobile after palmitoylation. The (2)H quadrupolar splittings allow the conformation of the ethanolamine group in acyl-gA to be determined. By combining our data with the previously determined quadrupolar splittings for deuterium labels on the palmitoyl chain [Vogt, T.C.B., Killian, J.A., & de Kruijff, B. (1994) Biochemistry 33, 2063-2070], we also propose a model for the acyl chain. The ethanolamine group rotates over Leu(10) and toward the outside of the gA channel's cylinder upon acylation, so that the attached acyl chain passes between the side chains of Trp(9) and Leu(10). To accommodate the acyl chain, the six-membered portion of the indole ring of Trp(9) is displaced by about 0.9 angstroms, by means of 1-2 degree rotations in chi(1) and chi(2).

Gramicidin↗

PubMed1996

Induction of nonbilayer structures in diacylphosphatidylcholine model membranes by transmembrane alpha-helical peptides: importance of hydrophobic mismatch and proposed role of tryptophans.

We have investigated the effect of several hydrophobic polypeptides on the phase behavior of diacylphosphatidylcholines with different acyl chain length. The polypeptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked. First it was demonstrated by circular dichroism measurements that these peptides adopt an alpha-helical conformation with a transmembrane orientation in bilayers of dimyristoylphosphatidylcholine. Subsequent 31P NMR measurements showed that the peptides can affect lipid organization depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. When a 17 amino acid residue long peptide (WALP17) was incorporated in a 1/10 molar ratio of peptide to lipid, a bilayer was maintained in saturated phospholipids containing acyl chains of 12 and 14 C atoms, an isotropic phase was formed at 16 C atoms, and an inverted hexagonal (HII) phase at 18 and 20 C atoms. For a 19 amino acid residue long peptide (WALP19) similar changes in lipid phase behavior were observed, but at acyl chain lengths of 2 C-atoms longer. Also in several cis-unsaturated phosphatidylcholine model membranes it was found that these peptides and a shorter analog (WALP16) induce the formation of nonbilayer structures as a consequence of hydrophobic mismatch. It is proposed that this unique ability of the peptides to induce nonbilayer structures in phosphatidylcholine model membranes is due to the presence of two tryptophans at both sides of the membrane/water interface, which prevent the peptide from aggregating when the mismatch is increased. Comparison of the hydrophobic length of the bilayers with the length of the different peptides showed that it is the precise extent of mismatch that determines whether the preferred lipid organization is a bilayer, isotropic phase, or HII phase. The peptide-containing bilayer and HII phase were further characterized after sucrose density gradient centrifugation of mixtures of WALP16 and dioleoylphosphatidylcholine. 31P NMR measurements of the isolated fractions showed that a complete separation of both components was obtained. Chemical analysis of these fractions in samples with varying peptide concentration indicated that the HII phase is highly enriched in peptide (peptide/lipid molar ratio of 1/6), while the maximum solubility of the peptide in the lipid bilayer is about 1/24 (peptide/lipid, molar). A molecular model of the peptide-induced HII phase is presented that is consistent with the results obtained thus far.

Amino Acid Sequence↗

PubMed1996

Engineering the gramicidin channel.

The chemical design or redesign of proteins with significant biological activity presents formidable challenges. Ion channels offer advantages for such design studies because one can examine the function of single molecular entities in real time. Gramicidin channels are attractive for study because of their known structure and exceptionally well-defined function. This article focuses on amino acid sequence changes that redesign the structure or function of gramicidin channels. New, and functional, folded states have been achieved. In some cases, a single amino acid sequence can give rise to several (up to three) functional conformations. Single amino acid substitutions confer voltage-dependent channel gating. The findings provide insight into the folding of integral membrane proteins, the importance of tryptophan residues at the membrane/water interface, and the mechanism of channel gating.

Amino Acid Sequence↗

PubMed1995

Gramicidin channel function does not depend on phospholipid chirality.

Chiral interactions are often important determinants for molecular recognition in chemistry and biochemistry. In order to determine whether the phospholipid backbone could be important for the conformational preference of membrane-spanning channels, we made use of the linear pentadecapeptide antibiotic gramicidin A (gA+) and a Trp-->Phe-substituted gA+ analogue, gramicidin M+ (gM+), as well as their enantiomers [gramicidin A- (gA-) and gramicidin M- (gM-), respectively]. All four analogues form conducting channels in planar bilayers formed from the dialkylphospholipids (R)- or (S)- dioleylphosphatidylcholine or from the diacylphospholipid (R)-dioleoylphosphatidylcholine. The characteristics of channels formed by the two gramicidin A enantiomers, or the two gramicidin M enantiomers, in membranes formed by either of the dioleylphosphatidylcholine enantiomers are indistinguishable. Similarly, channels formed by either pair of gramicidin enantiomers in dioleoylphosphatidylcholine bilayers are indistinguishable. We conclude that chiral interactions between gramicidin channels and the lipids in the host bilayer cannot be important determinants of gramicidin channel structure or function. The membrane/solution interface, therefore, seems to organize the channel structure because of the general characteristics of the nonpolar/polar transition at the interface rather than because of specific chemical interactions.

Amino Acid Sequence↗

PubMed1995

Palmitoylation-induced conformational changes of specific side chains in the gramicidin transmembrane channel.

To gain insight into the structural consequences of acylation for membrane proteins, we have covalently attached palmitic acid to the ethanolamine end of gramicidin A (gA), which functions as a well-characterized cation-selective membrane channel. Next, we investigated by NMR methods the effect of acylation on the side chains of Trp9, Leu10, and Trp11, which are expected to be close to the acyl chain, and of Val7, which is expected to be far from the acyl chain. Two-dimensional NMR spectroscopy in a sodium dodecyl sulfate (SDS) environment suggests that one of the beta-hydrogens of Leu10 of gA is severely shielded by a nearby aromatic ring. This shielding disappears upon acylation. Deuterium NMR spectra for labeled samples in hydrated dimyristoylphosphatidylcholine (DMPC) bilayers show that, for the major gA conformation, the (deuterated) side chains of Trp9 and Leu10 are markedly influenced by acylation, whereas the side chains of Val7 and Trp11 are essentially unaffected. The NMR results in both environments suggest that the indole ring of Trp9 is situated near the side chain of Leu10 and moves away upon acylation. We propose that acylation provides a subtle mechanism to modulate protein and lipid interactions and to regulate the stability and function of proteins within membranes.

Acylation↗

PubMed1995

Stabilizing effect of D-alanine2 in gramicidin channels.

We have investigated the effects of replacing Gly2 by D-Ala2 in gramicidin A (gA) analogues that have either L-Val, L-Ala, or Gly as the formyl-N-terminal residue. Circular dichroism, two-dimensional nuclear magnetic resonance, and hybrid channel experiments all show that [Ala1,D-Ala2]gA channels are structurally equivalent to the native [Val1,Gly2]gA channels, being formyl-NH-to-formyl-NH dimers of single-stranded, right-handed beta 6.3 helices. Replacing the Val1 of gA by Ala or Gly decreases the average channel duration. Replacing Gly2 by D-Ala in [Val1,Gly2]gA increases the average channel duration 4-fold and the single-channel conductance by approximately 15%; replacing Gly2 with D-Ala in [Ala1,Gly2]gA or [Gly1,Gly2]-gA leads in each case to a 10-fold increase in the average channel duration with only modest changes in the single-channel conductance, which depends on the identity of the position-one residue and the permeant ion. These results illustrate the importance of neighboring-residue side chain and backbone interactions for the modulation of channel properties.

Alanine↗

PubMed1995

Role of lysine-195 in the KMSKS sequence of E. coli tryptophanyl-tRNA synthetase.

Lysine 195 in the K195 MSKS sequence of E. coli tryptophanyl-tRNA synthetase (TrpRS) was replaced with alanine. The resulting K195A mutant TrpRS had essentially unchanged Km values for ATP and Trp, but a 1500-fold decreased kcat in a pyrophosphate-ATP exchange reaction. This large decrease in kcat reduces the rate of aminoacyladenylate formation (step 1) to a rate comparable to the rate of aminoacylation of tRNA(Trp) (step 2) by the K195A mutant enzyme. Both the TIGN and KMSKS sequences are important for step 1 of class I aminoacyl-tRNA synthetase reactions.

Adenosine Triphosphate↗

PubMed1995

Voltage-dependent gating of an asymmetric gramicidin channel.

In an effort to understand the molecular mechanisms of voltage activation of ion channels, we have chosen a system of known structure and examined the properties of heterodimeric channels formed between [Val1]gramicidin A ([Val1]gA) and [F6Val1]gramicidin A ([F6Val1]gA). Gramicidin channels are usually not voltage-dependent; but the introduction of a single symmetry-breaking dipolar F6Val1 residue into a ([Val1]gA)2 dimer to form the [F6Val1]gA/[Val1]gA heterodimer induces voltage-dependent transitions between two conducting states: a high-conductance state and a zero conductance (closed) state. The distribution between these states varies as a function of the applied potential but is not dependent on the nature of the permeant ion (H+ or Cs+). The permeating ions do not seem to contribute to the apparent gating charge.

Calcium Channels↗

PubMed1994

Helix sense of gramicidin channels as a "nonlocal" function of the primary sequence.

Gramicidin A (gA) channels are dimers formed by right-handed beta 6.3-helical monomers. The stereochemical basis for the preference of a right-handed conformation remains obscure, but it has earlier been demonstrated that the handedness can be shifted by changing the chirality of each residue in the LD-sequence and therefore is determined by the peptide itself and not by channel-membrane interactions. We now examine the contributions of Trp15, the central Val residues 6-8, and residues 1-5. None of these alone are sufficient to specify the helix sense. To examine the D-Val6-L-Val7-D-Val8 sequence, the register of the 3 valines was shifted by one to L-Val5-D-Val6-L-Val7. The resulting analogue, [Val5,D-Ala8]gA, forms channels with a conductance and duration that are both somewhat less than those of gA channels. The reduced channel duration can be attributed to a steric conflict between the side chains of Val1 in one monomer and Val5 in the other monomer. The helix handedness is not altered by this modification, as shown by circular dichroism and two-dimensional nuclear magnetic resonance spectroscopy and by hybrid channel experiments. [Val5,D-Ala8]gA forms hybrid channels with gA (which forms right-handed channels), but not with des-Val1-gA- (which forms left-handed channels). Similar hybrid channel analysis shows that des-Trp15-gA and [L-Ala1,D-Ala2,L-Ala3,D-Ala4]gA also form right-handed channels. We conclude that the helix handedness most probably is a complex function of the arrangement of both the D-Val-L-Val-D-Val and the L-Trp-(D-Leu-L-Trp)3 segments.

Amino Acid Sequence↗

PubMed1994

Role of the TIGN sequence in E. coli tryptophanyl-tRNA synthetase.

Tryptophanyl-tRNA synthetase in E. coli does not have the HIGH sequence that is normally characteristic of class I aminoacyl-tRNA synthetases (EC 6.1.1.2), but instead contains a TIGN sequence at residues 17-20, which has been suggested to be equivalent to the HIGH sequence (Jones, M.D. et al. (1986) Biochemistry 25, 1887-1891). We have overexpressed E. coli Trp-tRNA synthetase and have used site-directed mutagenesis to mutate Thr-17 in the TIGN sequence to alanine. The mutant enzyme has the same Km values as the wild-type for tryptophan or tRNA(Trp), and a slightly increased Km for ATP, from 0.37 to 0.64 mM. On the other hand, the kcat for either the first step or the overall reaction is decreased by a factor of 30. In comparing the Thr-17 and Ala-17 enzymes, the delta delta G for the conversion of substrate to transition state is +9.6 kJ/mol (2.3 kcal/mol). Thr-17 is therefore important in binding the substrate in the transition state, thus supporting the suggestion that TIGN may fulfill the role of a HIGH sequence.

Amino Acid Sequence↗

PubMed1994

Gramicidin A/short-chain phospholipid dispersions: chain length dependence of gramicidin conformation and lipid organization.

Gramicidin-lipid interactions were investigated using diacylphosphatidylcholines that contained two identical acyl chains of varying length, between 6 and 14 carbons. The gramicidin A (gA) conformation was monitored by circular dichroism (CD) spectroscopy and high-performance size-exclusion chromatography, and the lipid organization was investigated using 31P and 1H NMR spectroscopy and negative-stain electron microscopy. Diacylphosphatidylcholine (PC) lipids with chain lengths between 4 and 8 carbons have been previously shown to have a micellar organization in aqueous solution [Lin, T.-L., et al. (1986) J. Am. Chem. Soc. 108, 3499-3507]. CD spectra of aqueous gA/lipid dispersions, at a ratio of 1:28, demonstrated that the channel conformation of gA can be readily obtained when the acyl chain length is > or = 10, but not when the chain length is < or = 7. Size-exclusion chromatography revealed that the fraction of gA that could easily be dissociated into monomers in the dispersions increased with increasing acyl chain length, in agreement with the CD results. For a chain length of 8, the results were intermediate. The formation of the channel structure was found to depend on the "solvent-history", the temperature, the gA and lipid concentrations, the gA:lipid ratio, and consequently on the method of sample preparation. 1H and 31P NMR results suggest that codispersed gA increases the size of dioctanoyl-PC aggregates, but not of dihexanoyl-PC micelles. Negative-stain electron microscopy directly supports these findings. Dihexanoyl-PC (28 mM) was able to solubilize 1 mM gA in H2O, but the gA was not in the "channel" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Closed state of gramicidin channel detected by X-ray in-plane scattering.

An analogue of gramicidin A (gA) was synthesized with the formyl group replaced by a BOC group. The analogue (BOC-gA) exhibited single channel conduction, but the channel is 5-order-of-magnitude destabilized relative to the gA channel. Hydrated mixtures of gramicidin and dilauroyl phosphatidylcholine in the molar ratio of 1:10 were prepared into uniformly aligned multiple bilayers, and X-ray scattering with the momentum transfer in the plane of the membrane was measured. Analysis with the help of computer simulations showed that 70% of BOC-gA are monomers. Thus for the first time it was shown that gramicidin monomers are stable inside the monolayers of a lipid membrane. Furthermore, the monomers have the same beta helical conformation as the dimeric channel. The result suggests the possibility that when a gramicidin channel is closed, it dissociates into two monomers floating in opposite monolayers.

Computer Simulation↗

PubMed1994

Orientations of the tryptophan 9 and 11 side chains of the gramicidin channel based on deuterium nuclear magnetic resonance spectroscopy.

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp9 and Trp11 in specific indole-d5-labeled samples of gramicidin A incorporated into dimyristoyl phosphatidylcholine bilayers in the beta 6.3 channel conformation. The magnitudes and signs of the deuterium quadrupolar splittings were fit to the rings and assigned to specific ring bonds, using a full rotation search of the chi 1 and chi 2 angles of each Trp and a least-squares method. Unique assignments were obtained. The data and assignments are in close agreement with four sets of (chi 1, chi 2) angles for each Trp in which the indole N-H is oriented toward the membrane's exterior surface. (Four additional sets of (chi 1, chi 2) angles with the N-H's pointing toward the membrane interior are inconsistent with previous observations.) One of the sets of (chi 1, chi 2) angles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramicidin in sodium dodecyl sulfate micelles. Together, the 1H and 2H nuclear magnetic resonance methods suggest that the Trp9 and Trp11 side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp11 could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp9 could be fit only under the assumption that the quadrupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between rings 11 and 9 is also consistent with the 15N data of Hu et al. (1993. Biochemistry. 32:7035-7047).

Amino Acid Sequence↗

PubMed1994

Asymmetric gramicidin channels: heterodimeric channels with a single F6Val1 residue.

Substitution of Val1 by 4,4,4,4',4',4'-F6Val in [Val1]gramicidin A ([Val1]gA) produces channels in which the effects of amino acid replacements on dimer stability and ion permeation are nonadditive. If only one Val1 (in a symmetric [Val1]gA channel) is substituted by F6Val, the resulting heterodimeric channels are destabilized relative to both homodimeric parent channels and the single-channel conductance of the heterodimeric channels is reduced relative to the parent channels (Russell, E. W. B., L. B. Weiss, F. I. Navetta, R. E. Koeppe II, and O. S. Andersen. 1986. Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position #1 in gramicidin A. Biophys. J. 49:673; Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. Energetics of gramicidin hybrid channel formation as a test for structural equivalence. Side-chain substitutions in the native sequence. J. Mol. Biol. 211:221-234). To understand the basis for this destabilization, we have examined further the characteristics of [F6Val1]/[Xxx1]gA heterodimers, where Xxx = Gly, Val, and Ala. These heterodimeric channels show rapid current transitions between (at least) two current levels and display asymmetric i-V characteristics. The orientation of the heterodimers relative to the applied potential was determined by asymmetric addition of the gramicidin analogs, one to each side of a preformed bilayer. The current transitions are most clearly illustrated for [F6Val1]/[Gly1]gA heterodimers, which possess two finite and well defined current levels. Based on the existence of these two conductance states and the analysis of duration and interval distributions, we conclude that the transitions between the two current levels correspond to conformational transitions in "stable" heterodimers. In the case of [F6Val1]/[Val1]gA and [F6Val1]/[Ala1]gA heterodimers, the low-conductance state is indistinguishable from zero. The two (or more) conductance states presumably correspond to different orientations of the dipolar F6Val1 side chain. The distribution between the high- and the low-conductance states varies as a function of potential in [F6Val1]/[Gly1]gA channels. These characteristics cause the [F6Val1]/nonpolar (Val, Ala, Gly)gA hybrid channels to serve as a "simple" model for understanding gating transitions in membrane-spanning channels.

Amino Acid Sequence↗

PubMed1993

Energetics of heterodimer formation among gramicidin analogues with an NH2-terminal addition or deletion. Consequences of missing a residue at the join in the channel.

We examined the properties of membrane-spanning channels formed by gramicidin analogues that differ from [Val1]gramicidin A by having a single residue deletion or insertion at the formyl-NH terminus and of hybrid channels formed between such 14-, 15-, and 16-residue analogues. The channels' backbone structure, and helix sense, are not affected by the sequence modifications, because hybrid channels were observed for all combinations tested, and there was no excess energetic cost associated with hybrid channel formation. When hybrid channels form between analogues of different length the hybrid channel stability depends on the nature of the sequence dissimilarity. If two analogues differ by one residue (delta n = 1), the hybrid channels are destabilized by approximately 10 kJ/mol, because there is a defect (a "gap" in the peptide backbone) at the join between the two beta 6.3-helical monomers such that the dimer is stabilized by only five intermolecular C = O ... H-N hydrogen bonds rather than the usual six. This defect also alters the hybrid channels' permeability characteristics: the single-channel conductances are decreased, as if there were an additional barrier to ion movement through the channel. If the formyl-NH-terminal residue is Gly (and delta n = 1), the hybrid channels show multi-state behavior with voltage-dependent transitions between two conductance levels. If two analogues differ by two residues (delta n = 2), the hybrid channels are stabilized by 3 kJ/mol, indicating that structural continuity at the join between the monomers has been restored, as have the hybrid channels' permeability characteristics. The increased hybrid channel stability (when delta n = 2) may arise from altered membrane-channel interactions.

Amino Acid Sequence↗

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↗