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The structure and function of biological membranes can be expected to be sensitive to the extent of hydrophobic matching between the length of the membrane-spanning part of intrinsic membrane proteins and the hydrophobic thickness of the lipid bilayer. To gain insight into the consequences of hydrophobic mismatch on a molecular level, we have carried out systematic studies on well-defined peptidehpid complexes, using artifical transmembrane peptides, anchored to the membrane by tryptophan residues. It is shown that hydrophobic mismatch can result in a dramatic change in lipid organization, and that the presence of interfacially localized aromatic amino acid residues is important for determining the exact consequences of hydrophobic mismatch.

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) provides a favorable host framework for investigations of the influence of guest amino acids, for example a third, centrally located, Trp residue, within the hydrophobic core of a well characterized transmembrane helix. It is crucial to note that the orientation and rotation of GWALP23 are sensitive to single-residue replacements, in part because the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We introduced a Trp residue into position 12 or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra of GWALP23-W12 reveal that the peptide remains helical and retains a dominant preferred tilted transmembrane orientation with only a low extent of dynamic averaging, comparable to GWALP23 itself. Indeed, the tilt and dynamics of GWALP23-W12 are quite similar to the values observed for GWALP23 in DMPC bilayer membranes. We have analyzed the dynamics of the peptide helices using a modified Gaussian treatment as well as a semi-static treatment. The results indicate that a central Trp residue at position 12 does not appreciably perturb the properties of bilayer-spanning GWALP23. (By contrast, Arg-12 or Lys-12, when charged, induces multi-state behavior for GWALP23 in bilayer membranes [PNAS 110, 1692].) Additionally, we are investigating the influence of cholesterol upon the properties of membrane-spanning GWALP23, GWALP23-W12 and GWALP23-K12.

Hydrophobic membrane-spanning helices often are flanked by interfacial aromatic or charged residues. In this paper, we compare the consequences of single Trp → Tyr substitutions at each interface for the properties of a defined transmembrane helix in the absence of charged residues. The choice of molecular framework is critical for these single-residue experiments because the presence of "too many" aromatic residues (more than one at either membrane-water interface) introduces excess dynamic averaging of solid state NMR observables. To this end, we compare the outcomes when changing W(5) or W(19), or both of them, to tyrosine in the well-characterized transmembrane peptide acetyl-GGALW(5)(LA)6LW(19)LAGA-amide ("GWALP23"). By means of solid-state (2)H and (15)N NMR experiments, we find that Y(19)GW(5)ALP23 displays similar magnitudes of peptide helix tilt as Y(5)GW(19)ALP23 and responds similarly to changes in bilayer thickness, from DLPC to DMPC to DOPC. The presence of Y(19) changes the azimuthal rotation angle ρ (about the helix axis) to a similar extent as Y(5), but in the opposite direction. When tyrosines are substituted for both tryptophans to yield GY(5,19)ALP23, the helix tilt angle is again of comparable magnitude, and furthermore, the preferred azimuthal rotation angle ρ is relatively unchanged from that of GW(5,19)ALP23. The extent of dynamic averaging increases marginally when Tyr replaces Trp. Yet, importantly, all members of the peptide family having single Tyr or Trp residues near each interface exhibit only moderate and not highly extensive dynamic averaging. The results provide important benchmarks for evaluating conformational and dynamic control of membrane protein function.

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).

Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that may serve as anchors to aid the stabilization of a tilted transmembrane orientation. The synthetic neutral peptide GWALP23 (acetyl-GG2ALWLALALALALALALWLAG22A-amide) with two interfacial Trp residues has proved to be surprisingly well-behaved with minimal dynamic averaging in a stable transmembrane orientation in lipid-bilayer membranes of varying thickness. To further investigate the effect of interfacial His residues, we have substituted G2 and G22 with histidine in HWALP23 (acetyl-GH2ALWLALALALALALALWLAH22A-amide). In addition, to explore the fraying or uncoiling of the ends of the peptide, we have incorporated 2H-Ala labels at positions A3 and A21 (underlined above), which are sensitive to helix integrity, outside the core region of the peptide, to compare the influence of interfacial residues on the extent of unwinding of the helix ends. Solid-state 2H NMR spectra of macroscopically aligned DOPC lipid bilayer samples and in the presence of 10% and 20% cholesterol confirmed that one or both helix ends are frayed in DOPC bilayers alone and in the presence of up to 20% cholesterol. To further understand the effects of histidine in transmembrane helices, we have also substituted W5 and W19 with histidine in GHALP23 (acetyl-GGALH5LALALALALALALH19LAGA-amide) with 2H-Ala labels at A3 and A21. “Geometric Analysis of Labeled Alanines” (GALA) shows the extent of coiling or unwinding at the terminals for HWALP23 and GHALP23. It is plausible that the helix fraying may be critical for the stability of the transmembrane helix orientation in lipid bilayer membranes.

To investigate His residues in lipid bilayer membranes, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as a favorable host peptide. Importantly, membrane-spanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra reveal a marked difference between the L12H mutant and the L12H-A13H double mutant. GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes, suggesting that the bilayer-incorporated histidine side chain is neutral (deprotonated) at an experimental pH of 5-6. By contrast, GWALP23-H12,13 is highly dynamic and exhibits multiple states which are in slow exchange on the NMR timescale. Indeed, the multi-state behavior of GWALP23-H12,13 between pH 4 and pH 9 resembles closely that of GWALP23-R12 (J. Am. Chem. Soc. 132, 5803). Further aspects of the pH dependence of transmembrane helices having one or two histidines are under investigation.

In recent years pathogenic bacteria have become increasingly resistant to common antibiotics, leading to a demand for new, more potent antibacterial drugs. The 25 amino acid peptide lactoferricin (LfB25) exhibits broad spectrum antimicrobial properties and is of interest for drug development. A cationic amidated hexapeptide, (LfB6: RRWQWR-NH2), containing three arginines and two tryptophans has been identified as the “antimicrobial active site.” Our lab has shown that the antimicrobial activity of LfB6 can be enhanced by methylation of the tryptophan indole nitrogen (MeTrp). After selective deuteration of the MeTrp residues, membrane interactions can be examined by solid-state 2H NMR spectroscopy in oriented bilayers composed of mammalian- and bacterial-like membranes. A symmetric heptapeptide with four arginines and two methyl-tryptophans (RRMeWQMeWRR-NH2; LfB7 MeTrp 3,5) was designed and found to increase antimicrobial activity relative to LfB6. 2H NMR spectra reveal that the MeTrp residues are aligned at the membrane surface, while 31P NMR spectra indicate very little perturbation to the lipid head groups. Partitioning assays in multilamellar vesicles demonstrate an increase in membrane binding correlated with percent anionic lipid, and Trp emission fluorescence spectroscopy confirms that the MeTrps are more water exposed in neutral compared to anionic lipid membranes. To investigate the importance of backbone flexibility for activity and membrane interaction, the central glutamine of the heptapeptide has been changed to either Gly or Pro. Initial results indicate that the Pro-constrained peptide, RRMeWPMeWRR, is less antimicrobial and has weaker membrane alignment and binding than when Gln is the central residue. We will present results for the more flexible peptide with Gly as the central residue, RRMeWGMeWRR.

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.

Peptides of the "WALP" family, acetyl-GWW(LA)(n)LWWA-[ethanol]amide, have proven to be opportune models for investigating lipid-peptide interactions. Because the average orientations and motional behavior of the N- and C-terminal Trp (W) residues differ, it is of interest to investigate how the positions of the tryptophans influence the properties of the membrane-incorporated peptides. To address this question, we synthesized acetyl-GGWW(LA)(n)-ethanolamide and acetyl-(AL)(n)WWG-ethanolamide, in which n = 4 or 8, which we designate as "N-anchored" and "C-anchored" peptides, respectively. Selected (2)H or (15)N labels were incorporated for solid-state nuclear magnetic resonance (NMR) spectroscopy. These peptides can be considered "half"-anchored WALP peptides, having only one pair of interfacial Trp residues near either the amino or the carboxyl terminus. The hydrophobic lengths of the (n = 8) peptides are similar to that of WALP23. These longer half-anchored WALP peptides incorporate into lipid bilayers as α-helices, as reflected in their circular dichroism spectra. Solid-state NMR experiments indicate that the longer peptide helices assume defined transmembrane orientations with small non-zero average tilt angles and moderate to high dynamic averaging in bilayer membranes of 1,2-dioleoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine, and 1,2-dilauroylphosphatidylcholine. The intrinsically small apparent tilt angles suggest that interactions of aromatic residues with lipid headgroups may play an important role in determining the magnitude of the peptide tilt in the bilayer membrane. The shorter (n = 4) peptides, in stark contrast to the longer peptides, display NMR spectra that are characteristic of greatly reduced motional averaging, probably because of peptide aggregation in the bilayer environment, and CD spectra that are characteristic of β-structure.

By using selected (2)H and (15)N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) J. Biol. Chem. 285, 31723-31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW(5)LALALAP(12)ALALALW(19)LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed (2)H and (15)N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2)H GALA and (15)N-(1)H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ~34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ~30 ± 5°, with an apparent helix unwinding or "swivel" angle of ~70°. In DLPC and DOPC, on the basis of (2)H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala(21) in the phospholipids DMPC and DLPC yet remains intact through Ala(21) in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues.

Synthetic model peptides have proven useful for examining fundamental peptide-lipid interactions. A frequently employed peptide design consists of a hydrophobic core of Leu-Ala residues with polar or aromatic amino acids flanking each side at the interfacial positions, which serve to "anchor" a specific transmembrane orientation. For example, WALP family peptides (acetyl-GWW(LA)(n)LWWA-[ethanol]amide), anchored by four Trp residues, have received particular attention in both experimental and theoretical studies. A recent modification proved successful in reducing the number of Trp anchors to only one near each end of the peptide. The resulting GWALP23 (acetyl-GGALW(5)(LA)(6)LW(19)LAGA-[ethanol]amide) displays reduced dynamics and greater sensitivity to lipid-peptide hydrophobic mismatch than traditional WALP peptides. We have further modified GWALP23 to incorporate a single tyrosine, replacing W(5) with Y(5). The resulting peptide, Y(5)GWALP23 (acetyl-GGALY(5)(LA)(6)LW(19)LAGA-amide), has a single Trp residue that is sensitive to fluorescence experiments. By incorporating specific (2)H and (15)N labels in the core sequence of Y(5)GWALP23, we were able to use solid-state NMR spectroscopy to examine the peptide orientation in hydrated lipid bilayer membranes. The peptide orients well in membranes and gives well-defined (2)H quadrupolar splittings and (15)N/(1)H dipolar couplings throughout the core helical sequence between the aromatic residues. The substitution of Y(5) for W(5) has remarkably little influence on the tilt or dynamics of GWALP23 in bilayer membranes of the phospholipids DOPC, DMPC, or DLPC. A second analogue of the peptide with one Trp and two Tyr anchors, Y(4,5)GWALP23, is generally less responsive to the bilayer thickness and exhibits lower apparent tilt angles with evidence of more extensive dynamics. In general, the peptide behavior with multiple Tyr anchors appears to be quite similar to the situation when multiple Trp anchors are present, as in the original WALP series of model peptides.

Designed α-helical peptides such as GWALP23 serve as useful models for probing the influence of polar amino acids within a core transmembrane helical sequence. We incorporated lysine as a guest residue into the membrane-spanning host peptides GWALP23 and closely related Y5GWALP23 (acetyl-GGAL(W5/Y5)LALALAL12AL14ALALW19LAGA-amide). Lysine was introduced at either position 12 or 14 of the host sequences, for which position 12 corresponds to the center. Solid-state NMR spectra of 2H-Ala residues in peptides incorporated into oriented lipid bilayer samples reveal that L14K mutant peptides adopt well-defined orientations in DOPC, DMPC and DLPC. In each lipid membrane, the L14K substitution increases the helix tilt at neutral pH. The L12K substitution, on the other hand, reduces the 2H NMR spectral quality at neutral pH, particularly in the thicker DOPC, suggesting a lack of distinct orientation, as the system struggles to insert a charged lysine into the thicker bilayers. As the positively charged K12 amino group is titrated to higher pH values, nevertheless, the 2H NMR spectral quality improves in DOPC, and the K12 peptides adopt an average orientation nearly matching the one found for both host peptides GWALP23 and Y5GWALP23 (with L12). In similar fashion, titration of K14, in any of the tested lipid bilayer membranes, results in a smaller helix tilt, again much closer to that observed for the L14 peptides without a polar guest residue. Steady-state fluorescence measurements using the Y5GWALP23 series of peptides reveal spectral narrowing and modest blue shifts in Δmax from the W19 reporter, when either K12 or K14 is rendered non-ionized, suggesting a somewhat more hydrophobic environment for the Trp indole ring when the guest Lys side chain is neutral.

Aromatic or polar amino acid residues often flank each side of a membrane-spanning alpha helix, whether in an integral membrane protein or a model peptide. The aromatic residues Trp, Tyr and to a lesser extent Phe tend to partition to the membrane-water interface and act as “anchors” to stabilize the transmembrane orientation. The synthetic model peptide, GWALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide), has proven valuable for experimentation, with only one Trp “anchor” near each end of the transmembrane sequence. Indeed, with relatively minimal complications from the peptide dynamics, the average tilt of GWALP23 has been shown to vary systematically in lipid bilayer membranes of different thickness (see J. Biol. Chem. 285, 31723). We have employed 2H-alanines and solid-state NMR spectroscopy to investigate the consequences of moving or replacing W5 or W19 in GWALP23 with selected Tyr, Phe or Trp residues at the same or nearby locations. We find that GWALP23 peptides having Y5, F5 or W5 exhibit essentially the same average tilt in bilayer membranes of DOPC, DMPC or DLPC; with somewhat increased dynamics for the F5 peptide. When “double” anchors are present in Y4,5GWALP23 or F4,5GWALP23, the peptides appear less responsive to the bilayer thickness, as the dynamics become dramatically more extensive. Moving W19 to position 18, a 100° radial change, alters the direction of the helix tilt, as expected. We conclude that, in the absence of other functional groups, the aromatic residues determine the preferred orientations and dynamics of transmembrane peptides. Increased dynamics are observed when the ring hydrogen bonding is removed (Phe), or when two aromatic anchors are present on one side of the core transmembrane 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.

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 β 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.

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).

Linear rate-equilibrium (RE) relations, also known as linear free energy relations, are widely observed in chemical reactions, including protein folding, enzymatic catalysis, and channel gating. Despite the widespread occurrence of linear RE relations, the principles underlying the linear relation between changes in activation and equilibrium energy in macromolecular reactions remain enigmatic. When examining amphiphile regulation of gramicidin channel gating in lipid bilayers, we noted that the gating process could be described by a linear RE relation with a simple geometric interpretation. This description is possible because the gating process provides a well-understood reaction, in which structural changes in a bilayer-embedded model protein can be studied at the single-molecule level. It is thus possible to obtain quantitative information about the energetics of the reaction transition state and its position on a spatial coordinate. It turns out that the linear RE relation for the gramicidin monomer-dimer reaction can be understood, and the quantitative relation between changes in activation energy and equilibrium energy can be interpreted, by considering the effects of amphiphiles on the changes in bilayer elastic energy associated with channel gating. We are not aware that a similar simple mechanistic explanation of a linear RE relation has been provided for a chemical reaction in a macromolecule. RE relations generally should be useful for examining how amphiphile-induced changes in bilayer properties modulate membrane protein folding and function, and for distinguishing between direct (e.g., due to binding) and indirect (bilayer-mediated) effects.

We investigated the effects of substituting two of the four tryptophans (the "inner pair" Trp(9) and Trp(11) or the "outer pair" Trp(13) and Trp(15)) in gramicidin A (gA) channels. The conformational preferences of the doubly substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA's conformational preference in lipid bilayer membranes. [Phe(13,15)]gA largely retains the single-stranded helical channel structure, whereas [Phe(9,11)]gA exists primarily as double-stranded 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 Phe-substituted 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 are ~50% and lifetimes that are ~80% of 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% of 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 single-channel conductance than are the dipoles of Trp(13) and Trp(15).

This article summarizes methods for the chemical synthesis and biophysical characterization of gramicidins with varying sequences and labels. The family of gramicidin channels has developed into a powerful model system for understanding fundamental properties, interactions, and dynamics of proteins and lipids generally, and ion channels specifically, in biological membranes.

Lactoferricin B is a cationic antimicrobial peptide with broad spectrum effectiveness. A small piece extracted from this peptide, LfB6 (RRWQWR-NH2), has similar antimicrobial properties, which can be further enhanced by attaching a short fatty acid to the N-terminus (C6-LfB6). The exact mechanism by which antimicrobial peptides interact with bacterial cell membranes is not well understood, but it is proposed to depend on lipid composition. In contrast to mammalian membranes which are comprised primarily of neutral lipids, bacterial membranes contain a significant (∼20-25%) fraction of negatively charged lipids. In the case of LfB6, the presence of two tryptophans and three arginines are thought to promote selective interaction with bacterial cell membranes. Here, we investigate the interactions of C6-LfB6 with lipid bilayers by combining solid state 2H and 31P NMR with an ensemble of all-atom molecular dynamics simulations running in aggregate more than 10 microseconds. In particular, we have investigated the peptides interactions with bilayers with two distinct compositions: 3:1 POPE:POPG (bacteria-like) and POPC (mammal-like). The results show that at low concentration the peptide has very little effect on the acyl chain concentration of the anionic membrane, and a more substantial effect on the zwitterionic POPC membrane. The synergy between the experimental and simulation results generates new insights into the molecular-level physics driving antimicrobial function.

Peptides derived from lactoferricin B (LfB25; FKCRRWQWRMKKLGAPSITCVRRAF; +8), a 25-residue cationic innate immunity peptide released from bovine lactoferrin, exhibit broad spectrum antimicrobial and anti-angiogenic properties. An increase in drug-resistant bacteria and the role of angiogenesis in promoting tumor growth make LfB peptides attractive candidates for future drug development. An important principle for the design of peptide drugs is to reduce the number of amino acids and the sequence complexity, while maintaining maximal activity and minimal toxicity. LfB25 is proposed to inhibit angiogenesis, the formation of new blood vessels, by competing with fibroblast growth factor (FGF) for binding to negatively charged heparin sulfate proteoglycans on endothelial cell surfaces. Previously we used isothermal titration calorimetry (ITC) to characterize the binding of LfB25 and LfB6 (RRWQWR-NH2; +4; underlined above), the ‘antimicrobial core’ of LfB25, to heparin and sucrose octasulfate (SOS), a heparin analogue. The binding of LfB25 with SOS and heparin was found to fit a two site model, with Kd values on the order of 10−6 and 10−7; whereas the isotherms for LfB6 fit a single binding site model, with Kd values on the order of 10−6.We now report ITC binding assays for two new LfB peptides, LfB25-Ala3,20 and LfB11-Ala2,9 (KARRWQWRAKK-NH2; +7;sequence italicized above). To remove the disulfide bond, the two cysteines were replaced to give LfB25-Ala3,20. To reduce sequence length and complexity, in LfB11-Ala2,9 a cysteine and a methionine were changed to alanine, and the sequence was reduced to 11 residues. The binding of both LfB25-Ala3,20 and LfB11-Ala2,9 to SOS and heparin fit a single-site model, with Kd values on the order of 10−6, similar to those for LfB6. Results from antimicrobial and hemolytic assays will be presented.

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.

The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics of the membrane-spanning peptide. The results make up an important basis for understanding the functional role of proline in several families of membrane proteins.

With a pK > 12, the guanidinium side chain of arginine (Arg; R) is positively charged over a very wide pH range. There has been much recent discussion concerning the energetics of Arg inserting into a lipid bilayer, or Arg crossing a lipid bilayer. The topic holds significant intrinsic intellectual interest and also is important for understanding the gating mechanism of voltage-dependent transmembrane channels. We address this problem by direct experimental observation using a designed transmembrane peptide that has interfacial tryptophan (Trp; W) anchors. Within membrane-spanning, alpha-helical GWALP23, acetyl-GGALW5LALALAL12ALALALW19LAGA-amide (Vostrikov, et al. 2008. J Am Chem Soc 130, 12584), we have substituted either R12 or R14 near the putative helix midpoint. Models suggest that the W5 and W19 side chains project from essentially the same side of the GWALP23 alpha-helix, with R14 projecting from the opposite face and R12 from the same face as the W5 and W19 anchors. The R12 side chain in effect is situated between the W anchors. Based upon solid-state NMR spectra from oriented lipid/peptide samples, specifically the deuterium quadrupolar splittings from several 2H-labeled alanines in each Arg-containing peptide, the properties of these sequence isomers depend heavily upon the location of the Arg. We find that GWALP23-R14 adopts a transmembrane orientation with a tilt of about 17° in DOPC (compared to a tilt of about 6° for GWALP23 itself). By contrast, GWALP23-R12 seems to assume several different orientations with respect to a hydrated bilayer of DOPC; one or more of these orientations may represent surface-bound peptide.

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.

The transmembrane alpha helices of integral membrane proteins often contain flanking aromatic residues. If situated at or near the water-lipid membrane interface, these residues such as Trp may stabilize the transmembrane helices to their preferred orientation. In this work we investigate the influence of Trp anchor position on transmembrane peptide orientation and dynamics. To probe the effects of these aromatic residues, we have utilized GWALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide) as a model transmembrane peptide framework. We have incorporated specific 2H-labeled alanine residues within the core of the sequence for observation of peptide dynamics and orientation by means of solid-state NMR spectroscopy. GWALP23 contains two anchoring Trp residues at positions 5 and 19, and undergoes limited dynamic averaging of NMR observables, such as the quadrupolar splittings of 2H labeled alanine residues. Here, we monitor the relocation of Trp residues 5 and 19, 40° apart on the same helix face, to positions 4 and 20, which then are 200° apart and on opposite faces of the helix. In comparison with the host peptide GWALP23, many of the 2H quadrupolar splittings of Ala residues are changed in the GW(4,20)ALP23 peptide. For helices of GW(4,20)ALP23 in several lipid bilayers of different thickness, we are investigating whether the changes in the 2H-Ala quadrupolar splittings are due primarily to changes in helix orientation (tilt), azimuthal rotation about the helix axis, or dynamics, or perhaps a combination of factors, when the Trp residues are moved. We surmise that the locations of the tryptophans hold major influence over the mechanism by which these aromatic residues align themselves into favorable orientations at the water-lipid bilayer interface and help to govern the orientation and dynamics of the host transmembrane helix.

The rise in antibiotic-resistant strains of bacteria has led to an active search for more potent antimicrobial drugs. The hexapeptide (LfB6: RRWQWR-NH2) from the N-terminal of the protein lactoferricin was shown to be the antimicrobial ‘active core’ by Tomita (Acta Paediatr Jpn, 1994, 36:585). A heptapeptide synthesized in our lab, which contains 4 positively charged arginines and 2 methylated tryptophans (LfB7 MeTrp3,5: RRMeWQMeWRR-NH2), exhibits enhanced activity when compared to LfB6 against gram positive and gram negative bacteria. To determine if Trp-methylation is required for the increased efficacy, a control peptide without methylated Trp (LfB7: RRWQWRR-NH2) has been synthesized and characterized. In addition, glutamine was replaced with alanine in both peptides to examine the importance of H-bonding at the central position (LfB7 MeTrp3,5 Ala4: RRMeWAMeWRR-NH2and LfB7 Ala4: RRWAWRR-NH2). Replacement of the central Gln with Ala reduced activity 5-fold against E. coli. LfB7 MeTrp3,5 exhibits the highest activity, with a 4- and 6-fold increase compared to LfB7, against E. coli and S. aureus, respectively. The Ala, Trp and MeTrp residues were labeled with deuterium, and the peptide was studied by solid-state deuterium NMR in bacterial- and mammalian-like membranes. The 2H-quadrupolar splittings, whether from labeled Ala, MeTrp or Trp, are larger when MeTrp is present in the sequence, indicating a change in peptide conformation and/or dynamics upon Trp-methylation. Tryptophan emission fluorescence spectra reveal that the Trp and MeTrp residues are more deeply buried in anionic compared to neutral lipids, and circular dichroism spectra suggest that anionic lipids promote a conformational change that results in Trp-Trp (and MeTrp-MeTrp) interactions. These results emphasize the importance of Gln at the central position and of Trp-methylation for structure and activity of lactoferricin.

The study of charged residues in the hydrophobic interior of transmembrane proteins is crucial to the understanding of membrane proteins and their function. To this end, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide), a constructive low-dynamic model peptide, for investigations of single-residue influence on protein-lipid interactions (JBC 285, 31723). We have substituted a single Leu residue at position 12, within the hydrophobic core of the GWALP23 sequence, with either Arg (R12) or Glu (E12). Specific 2H-labeled Ala residues have been incorporated within the -R12 sequence for detection by solid-state 2H NMR. GWALP23-R12 remains charged in DOPC bilayers, even under strongly alkaline pH conditions, and displays multi-state behavior (JACS 132, 5803). To determine if the presence of an adjacent peptide with Glu at position 12 would influence the behavior of the -R12 peptide, we incorporate a mixture of labeled GWALP23-R12 and unlabeled -E12 peptides in DOPC bilayers. Preliminary results for oriented samples with both peptides suggest that -R12 remains multi-state in the presence of the -E12 peptide in DOPC bilayers at pH 6. By contrast, oriented samples of -R12 in DLPC bilayers show clearly defined quadrupolar splittings. In the presence of -E12, we observe small changes in the quadrupolar splittings, indicative of changes in orientation of the -R12 peptide. It is possible that peptide crowding within the bilayer may influence ionic interactions between the -E12 and -R12 peptides; therefore we are currently investigating the effect of varying the peptide/peptide and peptide/lipid ratios. Similar experiments with GW3,21ALP23-R12, with the Trp residues moved to the opposite face of the helix, also suggest interaction between -E12 and -R12 in both DLPC and DOPC bilayers at pH 6. Additionally, we are studying possible ion-pair interactions in a peptide with two ionizable residues, GWALP23-R12,E16.

GWALP23 (acetyl-GGALW5LALALAL12AL14AL16ALW19LAGA-amide) is a useful low-dynamic model peptide for investigations of single-residue influence on protein-lipid interactions and the properties of membrane-spanning helices (J. Biol. Chem. 285, 31723). To investigate the pH dependence, ionization behavior and orientational constraints when potentially negatively charged glutamic acid side chains are present, we have substituted a single Leu residue with Glu at different positions and have incorporated specific 2H-alanines in the core helix or near the ends (Ala-3 and Ala-21) of GWALP23. Solid-state NMR experiments show that the 2H-Ala quadrupolar splittings of core labels change very little over the pH range of 4 to 11.5 when E12, E14 or E16 is present in GWALP23, but show modest shifts above pH 12. The spectra from deuterium labels on alanines 3 and 21 show changes in the unwinding of helix terminals in all three mutants between pH 4.0 and 13. The combined results suggest that these Glu residues may titrate with a pKa near 12 in DLPC and DOPC lipid bilayers. The rather modest shifts in the core alanine 2H quadrupolar splittings, nevertheless, suggest that the orientation of the transmembrane helix actually may change rather little at high pH. Rather, the unwinding of helix ends may be a key indicator of Glu residue titration. Our results are consistent with the expectation that the buried negative charges will aggressively hold their waters of hydration. It is conceivable that the close proximity of E16 to the indole ring of W19 could influence the results.

Mutations and chemical substitutions of amino acid side chains and backbone atoms have proved vital for understanding the folding, structure and function of gramicidin channels in phospholipid membranes. The channel's pore is lined by peptide backbone groups; their importance for channel structure and function is shown by a single amide-to-ester replacement within the backbone, which greatly reduces the resulting channel conductance and lifetime. The four tryptophans and the intervening leucines together govern the formation and dissociation of conducting channels from single-stranded subunits. Conducting double-stranded gramicidin conformations (channels) occur rarely in membranes--except when the sequence has been altered to permit special arrangements of tryptophans or (infrequently) in unusually thick membranes. The tryptophans anchor the single-stranded channels to the membrane/solution interface, and the indole dipoles promote cation transport through the channels. Removal of any indole dipole reduces ion conductance; whereas 5-fluorination of an indole, which increases its dipole moment, enhances ion conductance. Some sequence changes at the formyl-NH-terminus (in the membrane interior, away from the tryptophans), including fluorination of the formyl-NH-terminal valine, introduce voltage-dependent channel gating. Gramicidin channels are not just static conductors, but also dynamic entities whose structure and function can be manipulated by backbone and side chain modifications.

The extent to which the length of the membrane-spanning part of intrinsic membrane proteins matches the hydrophobic thickness of the lipid bilayer may be an important factor in determining membrane structure and function. To gain insight into the consequences of hydrophobic mismatch on a molecular level, we have carried out systematic studies on well-defined peptide-lipid complexes. As model peptides we have chosen gramicidin A and a series of artificial hydrophobic alpha-helical transmembrane peptides that resemble the gramicidin channel. These peptides consist of a hydrophobic stretch of alternating leucine and alanine residues with variable length, flanked by tryptophan residues. Using wide-line NMR techniques, we have investigated the interaction of these peptides with the bilayer-forming diacyl phosphatidylcholines and with phospholipids which by themselves have a tendency to form non-bilayer structures. We have shown that hydrophobic mismatch leads to systematic changes of the bilayer thickness and that it can even change the macroscopic organization of the lipids. The type of lipid organization induced by the peptides and the efficiency of the various processes depend on the properties of the lipids and on the precise extent of mismatch.

The tryptophans of integral membrane proteins have been suggested to play specific roles as "interfacial anchors", based on their preference for a location near the lipid head groups. Still, the underlying mechanism behind this behavior remains unclear. NMR experiments can provide an important tool to study this interaction in an actual bilayer environment. Here solid-state deuterium nuclear magnetic resonance was used to study the tryptophans in membrane-spanning model peptides from the WALP family (acetyl-GWW(LA)nWWA-ethanolamide with n = 5 and 6.5) in samples of mechanically aligned dimyristoylphosphatidylcholine (DMPC) bilayers. The data indicate that the tryptophans near the C-terminal end of the peptide display a significantly different behavior from those near the N-terminus. This is reflected prominently in a large difference in the motion experienced by the indoles at either end of the peptide, highlighting the directionality of the helix. Nevertheless, our observations indicate high levels of motional freedom for all tryptophans in these membrane spanning domains that exceed the dynamics for the helix itself. These observations signify that steric and dynamic features of the polypeptide context modulate the tryptophan anchoring in the membrane interface. Measurements of WALP19 in the ether-linked DMPC analogue ditetradecylphosphatidylcholine (missing the lipid carbonyls) show very similar Trp dynamics and suggest similar orientations for some or all of the tryptophans. This suggests that the lipid acyl chain carbonyls play at most a minor role in the anchoring interaction between these Trp residues and the DMPC interfacial region.

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA- amide) is a constructive model peptide for investigations of single-residue effects on protein-lipid interactions and the properties of membrane-spanning helices (J.Biol. Chem. 285, 31723). GWALP23 has favorable properties in bilayer membranes because the peptide exhibits only limited dynamic averaging of NMR observables such as the 2H quadrupolar splitting or the 15N-1H dipolar coupling (Biophys. J. 101, 2939). To investigate the potential influence of negatively charged glutamic acid side chains upon system properties, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core of the single-Trp peptide Y5GWALP23 (see Biochemistry 51, 2044). Solid state 2H NMR experiments showed well defined 2H quadrupolar splittings for Y5GWALP23-E16 in the pH range from 4.0 to 9.0, suggesting that the peptide helix is well oriented in DOPC lipid bilayers. The E16-containing peptide seems to exhibit multi-state behavior at pH 10.9, in bilayers formed by ether-linked lipids, suggesting a pKa that is above pH 9 for the E16 side chain. The rather modest shift in the 2H quadrupolar splittings suggests that the orientation of the transmembrane peptide helix changes rather little at high pH. It is conceivable that the close proximity of E16 to W19 could provide stability to the neutral peptide helix and perhaps influence the pKa of E16. The molecular cousin having E14 instead of E16 shows multi-state behavior from pH 4.0 to pH 10.9 rendering pKa determination enigmatic at this time. Some puzzles remain with respect to comparisons between E16 and E14. We are additionally investigating the peptide-lipid behavior when Glu is introduced in position 12, at the peptide center.

We have used experimental deuterium NMR spectra from labeled tryptophans in membrane-spanning gramicidin A (gA)(1) channels to refine the geometry of the indole ring and, specifically, the C2-(2)H bond direction. By using partial exchange in a cold organic acid, we were able to selectively deuterate ring positions C2 and C5 and, thereby, define unambiguous spectral assignments. In a backbone-independent analysis, the assigned spectra from four distinct labeled tryptophans were used to assess the geometry of the planar indole ring. We found that the C2-(2)H bond makes an angle of about 6 degrees with respect to the normal to the indole ring bridge, and the experimental geometry was confirmed by density functional calculations using a 6-311G** basis set. The precisely determined ring geometry and the experimental spectra in turn are the foundation for calculations of the orientation of each tryptophan indole ring, with respect to the bilayer membrane normal, and of a principal order parameter S(zz) for each ring. The results have general significance for revising the tryptophan ring geometry that is used in protein molecular modeling, as well as for the analysis of tryptophan ring orientations in membrane-spanning proteins. The experimental precision in the definition of the indole ring geometry demonstrates yet another practical application emanating from fundamental research on the robust gramicidin channel.

To further investigate the effect of single amino acid substitution on the structure and function of the gramicidin channel, an analogue of gramicidin A (GA) has been synthesized in which Trp 1 5 is replaced by Gly in thecritical aqueous interface and cation binding region. The structure of Gly 1 5 -GA incorporated into SDS micelles has been determined using a combination of 2D-NMR spectroscopy and molecular modeling. Like the parent GA, Gly 1 5 -GA forms a dimeric channel composed of two single-stranded, right-handed β 6 , 3 -helices joined by hydrogen bonds between their N-termini. The replacement of Trp 1 5 by Gly does not have a significant effect on backbone structure or side chain conformations with the exception of Trp 1 1 in which the indole ring is rotated away from the channel axis. Measurement of the equilibrium binding constants and AG for the binding of monovalent cations to GA and Gly 1 5 -GA channels incorporated into PC vesicles using 2 0 5 Tl NMR spectroscopy shows that monovalent cations bind much more weakly to the Gly 1 5 -GA channel entrance than to GA channels. Utilizing the magnetization inversion transfer NMR technique, the transport of Na + ions through GA and Gly 1 5 -GA channels incorporated into PC/PG vesicles has been investigated. The Gly 1 5 substitution produces an increase in the activation enthalpy of transport and thus a significant decrease in the transport rate of the Na + ion is observed. The single-channel appearances show that the conducting channels have a single, well-defined structure. Consistent with the NMR results, the single-channel conductances are reduced by 30% and the lifetimes by 70%. It is concluded that the decrease in cation binding, transport, and conductance in Gly 1 5 -GA results from the removal of the Trp 1 5 dipole and, to a lesser extent, the change in orientation of Trp 1 1 .

Polyunsaturated fatty acids (PUFA) are found in great abundance in neural membranes where they are essential for function. Their incorporation into membrane phospholipids has also been proposed to be responsible, in part, for a plethora of health benefits associated with dietary consumption of fish oils. Extremely high disorder that deters close proximity to cholesterol is what distinguishes PUFA from more common monounsaturated and saturated fatty acids. The aversion to cholesterol is exemplified by a solubility of only ∼15 mol% in di-polyunsaturated 1,2-diarachidonylphosphatidylcholine (DAPC), as opposed to most phospholipid bilayers that can solubilize >50 mol%. According to our earlier neutron scattering experiments using deuterated analogs of cholesterol, the head group and tail of the sterol reside within the DAPC bilayer at the center. An orientation parallel to the plane of the bilayer and between leaflets is implied that, remarkably, differs from the generally accepted one in which the head group sits near the aqueous interface while the tail extends towards the middle of the bilayer. Here we present solid state 2H NMR spectra acquired with aligned multilayers that establish the bilayer normal constitutes the axis of motional averaging for a deuterated analog of cholesterol incorporated into DAPC. The dilemma that this result creates is being evaluated, including with the aid of molecular dynamics simulations.

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) is a constructive low-dynamic model peptide for investigations of single-residue influence on protein-lipid interactions and the properties of membrane-spanning helices (J. Biol. Chem. 285, 31723). To investigate the pH dependence, ionization behavior and orientational constraints when potentially negatively charged glutamic acid side chains are present, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core GWALP23 or Y5GWALP23. Solid state NMR experiments show well defined 2H-Ala quadrupolar splittings for Y5GWALP23-E14 over the pH range of 6 to 10, suggesting that the peptide helix is well oriented in DLPC and DOPC lipid bilayers. Spectral changes are evident above pH 11 (in ether-lipid bilayers), but the titration of the Glu is uncertain because the Tyr residue itself appears to titrate around pH 11.5. GWALP23-E14 shows no spectral changes over the pH range 6.0 to 11.5, yet a change in quadrupolar splittings is observed at pH 13. The combined results suggest that the Glu residue may titrate with a pKa near 12. In bilayers of DLPC and DOPC, GWALP23-E16 shows similar trend and is suggested to have a pKa of around 12. The rather modest shifts in the 2H quadrupolar splittings, nevertheless, suggest that the orientation of the transmembrane helix actually may change rather little at high pH. It is conceivable that the close proximity of either E14 or E16 to W19 could provide stability to the neutral peptide helix and perhaps influence the results. We additionally are investigating the possibilities for helix unwinding near the ends of these peptides.

Synthetic model peptides, such as GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide), provide a favorable “host” framework for investigations of the influence of chosen “guest” amino acids. For example, it is of interest to know the consequences of having a third, centrally located, tryptophan (Trp) within the hydrophobic core of a well characterized, anchored, transmembrane helix. It is crucial to note that the orientation and rotation of GWALP23 are sensitive to single-residue replacements, in part because the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). A Trp residue was introduced in the 12th or 13th position of GWALP23, and specific deuterated alanine labels (2H-Ala) were included as probes within the core helical sequence. The 2H quadrupolar splittings from solid-state NMR spectra of GWALP23-W12 and GWALP23-W13 show that the peptide remains helical and retains a dominant preferred tilted transmembrane orientation (similar to GWALP23) in lipid bilayer membranes of DOPC, DLPC, and DMPC. Modified Gaussian and semi-static treatments of the dynamics yield similar conclusions. While a central Trp at position 12 or 13 does not alter the characteristics of bilayer-spanning GWALP23, incorporation of the peptide helix into the bilayer membrane becomes more difficult. The properties of W4, 5 GWALP23 are also being investigated, for comparison with the highly dynamic Y4, 5 and the less dynamic F4, 5 peptides. Deuterium labels at Ala3 and Ala21 will allow assessment of possible fraying of the ends of selected helices in differing lipid membrane environments.

Transmembrane helices of integral membrane proteins often have aromatic residues flanking them. The aromatic residues favor locations within the membrane–water interface of a lipid bilayer and may serve as anchors to aid the stabilization of a tilted transmembrane orientation. To further understand the influence of Tyr, Trp or Phe residues upon the properties of helical membrane proteins (see Biochemistry 53, 3637), we have investigated the possibility of partial unwinding near the ends of selected transmembrane helices. For this purpose, we substituted positions 4 and 5 with either two Phe or two Ala residues to generate F4,5GWALP23 (acetyl-GGAF4F5(LA)6LW19LAGA-ethanolamide) or A4,5GWALP23 (acetyl-GGAA4A5(LA)6LW19LAGA-ethanolamide), respectively. By incorporating specific 2H-Ala labels at positions 3 and 21, we are able to compare the influence of interfacial Phe and Ala on the unwinding of the helix ends. Solid state NMR spectra of macroscopically aligned bilayer samples show well defined 2H-Ala quadrupolar splittings for both F4,5GWALP23 and A4,5GWALP23, suggesting that the peptide helices are well oriented in DLPC, DMPC and DOPC lipid bilayers. We are also able to estimate the helix tilt from deuterium labeling of the core alanine residues. Geometric Analysis of Labeled Alanines (GALA) then shows unwinding at the terminals for both F4,5GWALP23 and A4,5GWALP23. It is conceivable that the helix fraying may be critical for the stability of the transmembrane helix orientation in the lipid bilayer membranes.

In this study, we investigated the extent to which different aromatic and positively charged side chains, which often flank transmembrane segments of proteins, can influence lipid-peptide interactions. Model systems consisting of phosphatidylcholine and hydrophobic alpha-helical peptides with different flanking residues were investigated. The peptides were incorporated in relatively thick and in relatively thin lipid bilayers to create a peptide-bilayer hydrophobic mismatch, and the compensating effects on lipid structure were analyzed. When relatively long with respect to the thickness of the bilayer, the peptides that are flanked by the aromatic side chains, Trp, Tyr, and Phe, all induce a significant ordering of the lipid acyl chains, while the peptides flanked by the charged residues Lys, Arg, and His do not. However, when the peptides are relatively short with respect to the thickness of the bilayer, their effect on lipid organization does not depend primarily on their aromatic or charged character. Peptides flanked by Trp, Tyr, Lys, or (at low pH) His residues are effective in inducing mismatch-relieving cubic and inverted hexagonal phases, while analogues flanked by Phe, Arg, or (at neutral pH) His residues cannot induce an inverted hexagonal phase. The different responses to mismatch might reflect the different interfacial affinities of the residues that were investigated.

Bovine lactoferricin (LfB25) is a 25-residue peptide released from the N-terminal of bovine lactoferrin which exhibits broad spectrum antimicrobial properties. A hexapeptide, LfB6 (RRWQWR-NH2), is a small fragment that retains significant antimicrobial activity (Tomita et al. 1994. Acta Paediatr Jpn. 36:585). We seek to modify and understand the antimicrobial properties of LfB6 through various modifications. Previous studies from our lab have shown that antimicrobial activity can be increased by N-acylation and Trp-methylation (Greathouse et al. 2008. J. Pept. Sci. 14:1103), as well as by reversal of the peptide sequence (Retro-LfB6). Acylation and Trp-methylation enhance the binding of the peptides to bacterial-like membranes. In addition, Trp-methylation allows for selective ring labeling with deuterium for solid-state NMR studies. To determine whether combining the three modifications (sequence reversal, Trp methylation, and acylation) will further enhance activity, two Retro-LfB6 peptides have been synthesized with a methylated Trp at sequence position 4. One of the peptides has been further modified by N-acylation with a 6-carbon fatty acid. Retro-LfB6 (RWQMeWRR-NH2) and C6 Retro-LfB6 (C6-RWQMeWRR-NH2) were examined by solid-state 31P and 2H NMR in mechanically aligned bilayers composed of a neutral membrane with a zwitterionic head group (DMPC) and a bacterial-like membrane composed of a 3:1 mixture of neutral (DMPC) and anionic lipids (DMPG). 2H NMR spectra suggest weaker binding or less well-defined orientations of the Retro-LfB6 peptides compared to native LfB6 peptides. 31P NMR spectra reveal that the lipids remain primarily in a bilayer arrangement, with the peptides causing little change to the phosphate head groups. Antimicrobial assays demonstrate that the C6-acylated, Trp-methylated, Retro-LfB6 peptide has enhanced activity relative to the native peptide against S. aureus. Results from partitioning assays will be compared with the NMR and antimicrobial data.

Nano-electrospray ionization mass spectrometry (ESI-MS) was used to analyze hydrogen/deuterium (H/D) exchange properties of transmembrane peptides with varying length and composition. Synthetic transmembrane peptides were used with a general acetyl-GW(2)(LA)(n)LW(2)A-ethanolamine sequence. These peptides were incorporated in large unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The vesicles were diluted in buffered deuterium oxide, and the H/D exchange after different incubation times was directly analyzed by means of ESI-MS. First, the influence of the length of the hydrophobic Leu-Ala sequence on exchange behavior was investigated. It was shown that longer peptide analogs are more protected from H/D exchange than expected on the basis of their length with respect to bilayer thickness. This is explained by an increased protection from the bilayer environment, because of stretching of the lipid acyl chains and/or tilting of the longer peptides. Next, the role of the flanking tryptophan residues was investigated. The length of the transmembrane part that shows very slow H/D exchange was found to depend on the exact position of the tryptophans in the peptide sequence, suggesting that tryptophan acts as a strong determinant for positioning of proteins at the membrane/water interface. Finally, the influence of putative helix breakers was studied. It was shown that the presence of Pro in the transmembrane segment results in much higher exchange rates as compared with Gly or Leu, suggesting a destabilization of the alpha-helix. Tandem MS measurements suggested that the increased exchange takes place over the entire transmembrane segment. The results show that ESI-MS is a convenient technique to gain detailed insight into properties of peptides in lipid bilayers by monitoring H/D exchange kinetics.

The extent of matching of membrane hydrophobic thickness with the hydrophobic length of transmembrane protein segments potentially constitutes a major director of membrane organization. Therefore, the extent of mismatch that can be compensated, and the types of membrane rearrangements that result, can provide valuable insight into membrane functionality. In the present study, a large family of synthetic peptides and lipids is used to investigate a range of mismatch situations. Peptide conformation, orientation, and extent of incorporation are assessed by infrared spectroscopy, tryptophan fluorescence, circular dichroism, and sucrose gradient centrifugation. It is shown that peptide backbone structure is not significantly affected by mismatch, even when the extent of mismatch is large. Instead, this study demonstrates that for tryptophan-flanked peptides the dominant response of a membrane to large mismatch is that the extent of incorporation is reduced, when the peptide is both too short and too long. With increasing mismatch, a smaller fraction of peptide is incorporated into the lipid bilayer, and a larger fraction is present in extramembranous aggregates. Relatively long peptides that remain incorporated in the bilayer have a small tilt angle with respect to the membrane normal. The observed effects depend on the nature of the flanking residues: long tryptophan-flanked peptides do not associate well with thin bilayers, while equisized lysine-flanked peptides associate completely, thus supporting the notion that tryptophan and lysine interact differently with membrane-water interfaces. The different properties that aromatic and charged flanking residues impart on transmembrane protein segments are discussed in relation to protein incorporation in biological systems.

To better understand the mutual interactions between lipids and membrane-spanning peptides, we investigated the effects of tryptophan-anchored hydrophobic peptides of various lengths on the phase behavior of 1,2-dielaidoylphosphatidylethanolamine (DEPE) dispersions, using (31)P nuclear magnetic resonance and small-angle X-ray diffraction. Designed alpha-helical transmembrane peptides (WALPn peptides, with n being the total number of amino acids) with a hydrophobic sequence of leucine and alanine of varying length, bordered at both ends by two tryptophan membrane anchors, were used as model peptides and were effective at low concentrations in DEPE. Incorporation of 2 mol % of relatively short peptides (WALP14-17) lowered the inverted hexagonal phase transition temperature (T(H)) of DEPE, with an efficiency that seemed to be independent of the extent of hydrophobic mismatch. However, the tube diameter of the H(II) phase induced by the peptides was clearly dependent on mismatch and decreased with shorter peptide length. Longer peptides (WALP19-27) induced a cubic phase, both below and above T(H). Incorporation of WALP27, which is significantly longer than the DEPE bilayer thickness, did not stabilize the bilayer. The longest peptide used, WALP31, hardly affected the lipid's phase behavior, and appeared not to incorporate into the bilayer. The consequences of hydrophobic mismatch between peptides and lipids are therefore more dramatic with shorter peptides. The data allow us to suggest a detailed molecular model of the mechanism by which these transmembrane peptides can affect lipid phase behavior.

When the central valine residues 6, 7, and 8 of gramicidin A (gA) are shifted by one position, the resulting [Val(5), D-Ala(8)]gA forms right-handed channels with a single-channel conductance and average duration somewhat less than gA channels. The reduction in channel duration has been attributed to steric conflict between the side chains of Val(1) and Val(5) in opposing monomers (Koeppe, R. E. II, D. V. Greathouse, A. Jude, G. Saberwal, L. L. Providence, and O. S. Andersen. 1994. J. Biol. Chem. 269:12567-12576). To investigate the orientations and motions of valines in [Val(5), D-Ala(8)]gA, we have incorporated (2)H labels at Val 1, 5, or 7 and recorded (2)H-NMR spectra of oriented and nonoriented samples in hydrated dimyristoylphosphatidylcholine. Spectra of nonoriented samples at 4 degrees C reveal powder patterns that indicate rapid side chain "hopping" for Val(5), and an intermediate rate of hopping for Val(1) and Val(7) that is somewhat slower than in gA. Oriented samples of deuterated Val(1) and Val(7) show large changes in the methyl and C(beta)-(2)H quadrupolar splittings (Deltanu(q)) when Ala(5) in native gA is changed to Val(5). Three or more peaks for the Val(1) methyls with Deltanu(q) values that vary with the echo delay, together with an intermediate spectrum for nonoriented samples at 4 degrees C, suggest unusual side chain dynamics for Val(1) in [Val(5), D-Ala(8)]gA. These results are consistent with a steric conflict that has been introduced between the two opposing monomers. In contrast, the acylation of gA has little influence on the side chain dynamics of Val(1), regardless of the identity of residue 5.

This article summarizes methods for the chemical synthesis and biophysical characterization of gramicidins with varying sequences and labels. The family of gramicidin channels has developed into a powerful model system for understanding fundamental properties, interactions, and dynamics of proteins and lipids generally, and ion channels specifically, in biological membranes.

Specific interactions of membrane proteins with the membrane interfacial region potentially define protein position with respect to the lipid environment. We investigated the proposed roles of tryptophan and lysine side chains as "anchoring" residues of transmembrane proteins. Model systems were employed, consisting of phosphatidylcholine lipids and hydrophobic alpha-helical peptides, flanked either by tryptophans or lysines. Peptides were incorporated in bilayers of different thickness, and effects on lipid structure were analyzed. Induction of nonbilayer phases and also increases in bilayer thickness were observed that could be explained by a tendency of Trp as well as Lys residues to maintain interactions with the interfacial region. However, effects of the two peptides were remarkably different, indicating affinities of Trp and Lys for different sites at the interface. Our data support a model in which the Trp side chain has a specific affinity for a well defined site near the lipid carbonyl region, while the lysine side chain prefers to be located closer to the aqueous phase, near the lipid phosphate group. The information obtained in this study may further our understanding of the architecture of transmembrane proteins and may prove useful for refining prediction methods for transmembrane segments.