Single tryptophan and tyrosine comparisons in the N-terminal and C-terminal interface regions of transmembrane GWALP peptides.
ABSTRACT: 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 ? (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 ? 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.
Project description:Aromatic amino acids often flank the transmembrane alpha helices of integral membrane proteins. By favoring locations within the membrane-water interface of the lipid bilayer, aromatic residues Trp, Tyr, and sometimes Phe may serve as anchors to help stabilize a transmembrane orientation. In this work, we compare the influence of interfacial Trp, Tyr, or Phe residues upon the properties of tilted helical transmembrane peptides. For such comparisons, it has been critical to start with no more than one interfacial aromatic residue near each end of a transmembrane helix, for example, that of GWALP23 (acetyl-GGALW(5)(LA)6LW(19)LAGA-[ethanol]amide). To this end, we have employed (2)H-labeled 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 proximate locations. We find that GWALP23 peptides having F5, Y5, or W5 exhibit essentially the same average tilt and similar dynamics in bilayer membranes of 1,2-dilauroylphosphatidylcholine (DLPC) or 1,2-dioleoylphosphatidylcholine (DOPC). When double Tyr anchors are present, in Y(4,5)GWALP23 the NMR observables are markedly more subject to dynamic averaging and at the same time are less responsive to the bilayer thickness. Decreased dynamics are nevertheless observed when ring hydrogen bonding is removed, such that F(4,5)GWALP23 exhibits a similar extent of low dynamic averaging as GWALP23 itself. When F5 is the sole aromatic group in the N-interfacial region, the dynamic averaging is (only) slightly more extensive than with W5, Y5, or Y4 alone or with F4,5, yet it is much less than that observed for Y(4,5)GWALP23. Interestingly, moving Y5 to Y4 or W19 to W18, while retaining only one hydrogen-bond-capable aromatic ring at each interface, maintains the low level of dynamic averaging but alters the helix azimuthal rotation. The rotation change is about 40° for Y4 regardless of whether the host lipid bilayer is DLPC or DOPC. The rotational change (??) is more dramatic and more complex when W19 is moved to W18, as ?? is about +90° in DLPC but about -60° in DOPC. Possible reasons for this curious lipid-dependent helix rotation could include not only the separation distances between flanking aromatic or hydrophobic residues but also the absolute location of the W19 indole ring. For the more usual cases, when the helix azimuthal rotation shows little dependence on the host bilayer identity, excepting W(18)GWALP23, the transmembrane helices adapt to different lipids primarily by changing the magnitude of their tilt. We conclude that, in the absence of other functional groups, interfacial aromatic residues determine the preferred orientations and dynamics of membrane-spanning peptides. The results furthermore suggest possibilities for rotational and dynamic control of membrane protein function.
Project description:While the interfacial partitioning of charged or aromatic anchor residues may determine the preferred orientations of transmembrane peptide helices, the dependence of helix orientation on anchor residue position is not well understood. When anchor residue locations are changed systematically, some adaptations of the peptide-lipid interactions may be required to compensate for the altered interfacial interactions. Recently, we have developed a novel transmembrane peptide, termed GW(5,19)ALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide), which proves to be a well-behaved sequence for an orderly investigation of protein-lipid interactions. Its roughly symmetric nature allows for shifting the anchoring Trp residues by one Leu-Ala pair inward (GW(7,17)ALP23) or outward (GW(3,21)ALP23), thus providing fine adjustments of the formal distance between the tryptophan residues. With no other obvious anchoring features present, we postulate that the inter-Trp distance may be crucial for aspects of the peptide-lipid interaction. Importantly, the amino acid composition is identical for each of the resulting related GWALP23 sequences, and the radial separation between the pairs of Trp residues on each side of the transmembrane ?-helix remains similar. Here we address the adaptation of the aforementioned peptides to the varying Trp locations by means of solid-state (2)H nuclear magnetic resonance experiments in varying lipid bilayer membrane environments. All of the GW(x,y)ALP23 sequence isomers adopt transmembrane orientations in DOPC, DMPC, and DLPC environments, even when the Trp residues are quite closely spaced, in GW(7,17)ALP23. Furthermore, the dynamics for each peptide isomer are less extensive than for peptides possessing additional interfacial Trp residues. The helical secondary structure is maintained more strongly within the Trp-flanked core region than outside of the Trp boundaries. Deuterium-labeled tryptophan indole rings in the GW(x,y)ALP23 peptides provide additional insights into the behavior of the Trp side chains. A Trp side chain near the C-terminus adopts a different orientation and undergoes somewhat faster dynamics than a corresponding Trp side chain located an equivalent distance from the N-terminus. In contrast, as the inter-Trp distance changes, the variations among the average orientations of the Trp indole rings at either terminus are systematic yet fairly small. We conclude that subtle adjustments to the peptide tilt, and to the N- and C-terminal Trp side chain torsion angles, permit the GW(x,y)ALP23 peptides to maintain preferred transmembrane orientations while adapting to lipid bilayers with differing hydrophobic thicknesses.
Project description:Transmembrane domains of membrane proteins sometimes contain conserved charged or ionizable residues which may be essential for protein function and regulation. This work examines the molecular interactions of single Arg residues within a highly dynamic transmembrane peptide helix. To this end, we have modified the GW4,20ALP23 (acetyl-GGAW4(AL)7AW20AGA-amide) model peptide framework to incorporate Arg residues near the center of the peptide. Peptide helix formation, orientation and dynamics were analyzed by means of solid-state NMR spectroscopy to monitor specific 2H- or 15N-labeled residues. GW4,20ALP23 itself adopts a tilted orientation within lipid bilayer membranes. Nevertheless, the GW4,20ALP23 helix exhibits moderate to high dynamic averaging of NMR observables, such as 2H quadrupolar splittings or 15N-1H dipolar couplings, due to competition between the interfacial Trp residues on opposing helix faces. Here we examine how the helix dynamics are impacted by the introduction of a single Arg residue at position 12 or 14. Residue R14 restricts the helix to low dynamic averaging and a well-defined tilt that varies inversely with the lipid bilayer thickness. To compensate for the dominance of R14, the competing Trp residues cause partial unwinding of the helix at the C-terminal. By contrast, R12GW4,20ALP23 exits the DOPC bilayer to an interfacial surface-bound location. Interestingly, multiple orientations are exhibited by a single residue, Ala-9. Quadrupolar splittings generated by 2H-labeled residues A3, A5, A7, and A9 do not fit to the ?-helical quadrupolar wave plot defined by residues A11, A13, A15, A17, A19, and A21. The discontinuity at residue A9 implicates a helical swivel distortion and an apparent 310-helix involving the N-terminal residues preceding A11. These molecular features suggest that, while arginine residues are prominent factors controlling transmembrane helix dynamics, the influence of interfacial tryptophan residues cannot be ignored.
Project description:Both Trp and Arg in transmembrane protein domains make important interactions with lipids at the membrane/water interface, but at different depths. Derivatives of the designed peptide GWALP23, acetyl-GGALW(5)LALALALALALALW(19)LAGA-amide, with single Trp anchors, have proven useful for characterizing such interactions. Indeed, previous work revealed quite different effects emanating from Arg substitutions at positions 12 and 14 within GWALP23, with the R12 peptide exhibiting multiple positions and orientations with respect to DOPC bilayer membranes (Vostrikov et al. J. Am. Chem. Soc. 2010, 132, 5803-5811). To gain further understanding of the multistate behavior, we moved the Trp "anchor" residues to more outer positions 3 and 21 in GWALP23 itself, and in the R12 and R14 derivatives. The locations and orientations of the peptides with respect to lipid bilayer membranes of differing thickness were investigated by means of solid-state (2)H NMR spectroscopy, using labeled alanines, and coarse-grained molecular dynamics simulations. Interestingly, relatively intense and narrow (2)H resonances from selected backbone C(?) deuterons were observed over quite narrow ranges of frequency and sample orientation. The backbone resonances reflect dynamic complexities and at the same time provide important contributions for the analysis of peptide transmembrane orientation. With the Trp(3,21) anchors relatively far from the peptide and bilayer center, the results indicate significantly large apparent tilt angles, for example, close to 30° for the new R12 and R14 peptides with respect to the bilayer normal of DLPC membranes. The R12 side chain indeed is "rescued" to a stable position, where it is accommodated within the transmembrane helix, when the Trp anchors are moved outward and to another face of the helix. At the same time, the R14 side chain of transmembrane GW(3,21)ALP23 also retains a stable favored position.
Project description:In biological membranes the alignment of embedded proteins provides crucial structural information. The transmembrane (TM) parts have well-defined secondary structures, in most cases ?-helices and their orientation is given by a tilt angle and an azimuthal rotation angle around the main axis. The tilt angle is readily visualized and has been found to be functionally relevant. However, there exist no general concepts on the corresponding azimuthal rotation. Here, we show that TM helices prefer discrete rotation angles. They arise from a combination of intrinsic properties of the helix geometry plus the influence of the position and type of flanking residues at both ends of the hydrophobic core. The helical geometry gives rise to canonical azimuthal angles for which the side chains of residues from the two ends of the TM helix tend to have maximum or minimum immersion within the membrane. This affects the preferential position of residues that fall near hydrophobic/polar interfaces of the membrane, depending on their hydrophobicity and capacity to form specific anchoring interactions. On this basis, we can explain the orientation and dynamics of TM helices and make accurate predictions, which correspond well to the experimental values of several model peptides (including dimers), and TM segments of polytopic membrane proteins.
Project description: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 ?-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 ?-structure.
Project description: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.
Project description:Transmembrane ?-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock ?-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n?=?100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors.
Project description:The membrane-spanning segments of integral membrane proteins often are flanked by aromatic or charged amino acid residues, which may "anchor" the transmembrane orientation. Single spanning transmembrane peptides such as those of the WALP family, acetyl-GWW(LA)(n)LWWA-amide, furthermore adopt a moderate average tilt within lipid bilayer membranes. To understand the anchor residue dependence of the tilt, we introduce Leu-Ala "spacers" between paired anchors and in some cases replace the outer tryptophans. The resulting peptides, acetyl-GX(2)ALW(LA)(6)LWLAX(22)A-amide, have Trp, Lys, Arg, or Gly in the two X positions. The apparent average orientations of the core helical sequences were determined in oriented phosphatidylcholine bilayer membranes of varying thickness using solid-state (2)H NMR spectroscopy. When X is Lys, Arg, or Gly, the direction of the tilt is essentially constant in different lipids and presumably is dictated by the tryptophans (Trp(5) and Trp(19)) that flank the inner helical core. The Leu-Ala spacers are no longer helical. The magnitude of the apparent helix tilt furthermore scales nicely with the bilayer thickness except when X is Trp. When X is Trp, the direction of tilt is less well defined in each phosphatidylcholine bilayer and varies up to 70° among 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn-glycero-3-phosphocholine bilayer membranes. Indeed, the X = Trp case parallels earlier observations in which WALP family peptides having multiple Trp anchors show little dependence of the apparent tilt magnitude on bilayer thickness. The results shed new light on the interactions of arginine, lysine, tryptophan, and even glycine at lipid bilayer membrane interfaces.
Project description:Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central alpha-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with DeltaG degrees (H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.