Activity and structural comparisons of solution associating and monomeric channel-forming peptides derived from the glycine receptor m2 segment

A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor alpha(1) subunit (M2GlyR), including the 22-residue sequence NK(4)-M2GlyR p22 wild type (WT) (KKKKPARVGLGITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK(4)-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide adopts a bent conformation forming a hydrophobic pocket by hydrophobic interactions.     

High-resolution structural profile of hylaseptin-4: Aggregation,membrane topology and pH dependence of overall membrane binding process

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH₂) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d₃₈ micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.     

Folding determinants of disulfide bond forming protein B explored by solution nuclear magnetic resonance spectroscopy

The two-stage model for membrane protein folding postulates that individual helices form first and are subsequently packed against each other. To probe the two-stage model, the structures of peptides representing individual transmembrane helices of the disulfide bond forming protein B have been studied in trifluoroethanol solution as well as in detergent micelles using nuclear magnetic resonance (NMR) and circular dichroism spectroscopy. In TFE solution, peptides showed well-defined α-helical structures. Peptide structures in TFE were compared to the structures of full-length protein obtained by X-ray crystallography and NMR. The extent of α-helical secondary structure coincided well, lending support for the two-stage model for membrane protein folding. However, the conformation of some amino acid side chains differs between the structures of peptide and full-length protein. In micellar solution, the peptides also adopted a helical structure, albeit of reduced definition. Using measurements of paramagnetic relaxation enhancement, peptides were confirmed to be embedded in micelles. These observations may indicate that in the native protein, tertiary interactions additionally stabilize the secondary structure of the individual transmembrane helices.     

Structural and biophysical properties of a synthetic channel-forming peptide: Designing a clinically relevant anion selective pore

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK(4)-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Bilayer conformation of fusion peptide of influenza virus hemagglutinin: a molecular dynamics simulation study

Unraveling the conformation of membrane-bound viral fusion peptides is essential for understanding how those peptides destabilize the bilayer topology of lipids that is important for virus-cell membrane fusion. Here, molecular dynamics (MD) simulations were performed to investigate the conformation of the 20 amino acids long fusion peptide of influenza hemagglutinin of strain X31 bound to a dimyristoyl phosphatidylcholine (DMPC) bilayer. The simulations revealed that the peptide adopts a kinked conformation, in agreement with the NMR structures of a related peptide in detergent micelles. The peptide is located at the amphipathic interface between the headgroups and hydrocarbon chains of the lipid by an energetically favorable arrangement: The hydrophobic side chains of the peptides are embedded into the hydrophobic region and the hydrophilic side chains are in the headgroup region. The N-terminus of the peptide is localized close to the amphipathic interface. The molecular dynamics simulations also revealed that the peptide affects the surrounding bilayer structure. The average hydrophobic thickness of the lipid phase close to the N-terminus is reduced in comparison with the average hydrophobic thickness of a pure dimyristoyl phosphatidylcholine bilayer.     

Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor

The Ste2p receptor for alpha-factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form beta-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.     

Circular dichroism studies on a synthetic peptide corresponding to the membrane-spanning region of vesicular stomatitis virus G protein and its fatty acyl derivative

The conformations of synthetic peptides Lys-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-OCH3 and Lys(epsilon-palmitoyl)-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-O CH3, which constitute a part of the membrane-spanning region of the vesicular stomatitis virus G protein, have been studied by circular dichroism (CD) spectroscopy. Secondary structural features are observed for both peptides in trifluoroethanol, methanol, aqueous mixtures of trifluoroethanol and methanol and in a micellar environment. In trifluoroethanol, the CD spectra indicate the presence of a helical conformation, whereas in aqueous mixtures of organic solvents, both helical and beta-conformations are observed. While fatty acid acylation does not directly modulate peptide conformation, it promotes self-association of the acylated peptide and association with micelles. In a micellar environment, the acylated peptide adopts an alpha-helical conformation.     

Self-assembling of peptide/membrane complexes by atomistic molecular dynamics simulations

Model biological membranes consisting of peptide/lipid-bilayer complexes can nowadays be studied by classical molecular dynamics (MD) simulations at atomic detail. In most cases, the simulation starts with an assumed state of a peptide in a preformed bilayer, from which equilibrium configurations are difficult to obtain due to a relatively slow molecular diffusion. As an alternative, we propose an extension of reported work on the self-organization of unordered lipids into bilayers, consisting of including a peptide molecule in the initial random configuration to obtain a membrane-bound peptide simultaneous to the formation of the lipid bilayer. This strategy takes advantage of the fast reorganization of lipids, among themselves and around the peptide, in an aqueous environment. Model peptides of different hydrophobicity, CH3-CO-W2L18W2-NH2 (WL22) and CH3-CO-W2A18W2-NH2 (WA22), in dipalmitoyl-phosphatidylcholine (DPPC) are used as test cases. In the equilibrium states of the peptide/membrane complexes, achieved in time ranges of 50-100 ns, the two peptides behave as expected from experimental and theoretical studies. The strongly hydrophobic WL22 is inserted in a transmembrane configuration and the marginally apolar, alanine-based WA22 is found in two alternative states: transmembrane inserted or parallel to the membrane plane, embedded close to the bilayer interface, with similar stability. This shows that the spontaneous assembly of peptides and lipids is an unbiased and reliable strategy to produce and study models of equilibrated peptide/lipid complexes of unknown membrane-binding mode and topology.     

Homooligopeptides composed of hydrophobic amino acid residues interact in a specific manner by taking α-helix or β-structure toward lipid bilayers

In order to investigate the role of each amino acid residue in determining the secondary structure of the transmembrane segment of membrane proteins in a lipid bilayer, we made a conformational analysis by CD for lipid-soluble homooligopeptides, benzyloxycarbonyl-(Z-) Aaa_n-OEt (n = 5-7), composed of Ala, Leu, Val, and Phe, in three media of trifluoroethanol, sodium dodecyl sulfaie micelle, and phospholipid liposomes. The lipid-peptide interaction was also studied through the observation of bilayer phase transition by differential scanning cahrimetry (DSC). The CD studies showed that peptides except for Phe oligomers are present as a mainly random structure in trifluoroethanol, as a mixture of α-helix, β-sheet, β-turn, and /or random in micelles above the critical micellization concentration and preferably as an extended structure of α-helical or β-structure in dipalmitoyl-D,L -α-phosphatidylcholine (DPPC) liposomes of gel state. That the β-structure content of Val oligomers in lipid bilayers is much higher than that in micelles and the oligopeptides of Leu (n = 7) and Ala (n = 6) can take an α-helical structure with one to two turns in lipid bilayers despite their short chain lengths indicates that lipid bilayers can stabilize the extended structure of both α-helical and β-structures of the peptides. The DSC study for bilayer phase transition of DPPC / peptide mixtures showed that the Leu oligomer virtually affects neither the temperature nor the enthalpy of the transition, while Val and Ala oligomers slightly reduce the transition enthalpy without altering the transition temperature. In contrast, the Phe oligomer affects the phase transition in much more complicated manner. The decreasing tendency of the transition enthalpy was more pronounced for the Ala oligomer as compared with the Leu and Val oligomers, which means that the isopropyl group of the side chain has a less perturbing effect on the lipid acyl chain than the methyl group of Ala. © 1995 John Wiley & Sons, Inc.     

Solvent effects on the conformation of the transmembrane peptide gramicidin A: insights from electrospray ionization mass spectrometry

The binding of sodium ions to the transmembrane channel peptide gramicidin A has permitted the use of electrospray ionization mass spectrometry to study its conformation in different solvent environments. The mass spectra of the peptide in the various solvents suggest that different conformations of gramicidin A differ in their ability to bind metal ions. The data are consistent with monomeric behavior of gramicidin A in trifluoroethanol and dimethyl sulfoxide solutions, but reveal the presence of noncovalent intermolecular interactions in ethanol solution through the observation of heterodimers formed between the naturally occurring variants of the peptide. The addition of 50% v/v of water to the ethanolic solution causes changes in the circular dichroism spectrum of the peptide, suggestive of a shift in the equilibrium mixture of conformers present toward monomeric species, a result supported by its mass spectrum. The structure of gramicidin A in trifluoroethanol has also been investigated by hydrogen exchange measurements monitored by mass spectrometry. The observation of significant protection against exchange suggests that the monomeric peptide is highly structured in trifluoroethanol. The results indicate that mass spectrometry has the potential to probe the conformational behavior of neutral hydrophobic peptides in environments that mimic their functional states.     

The membrane-proximal tryptophan-rich region of the HIV glycoprotein, gp41, forms a well-defined helix in dodecylphosphocholine micelles

The membrane-proximal tryptophan-rich region of the HIV transmembrane glycoprotein, gp41, plays an important role in the membrane fusion reaction. Using NMR spectroscopy, we have studied the tertiary structure of a synthetic 19-residue amidated peptide (NH2-KWASLWNWFNITNWLWYIK-CONH2) corresponding to this region in membrane-mimetic environments. Initial experiments in sodium dodecyl sulfate/H2O micelles and trifluoroethanol gave poor results, because of low solubility. However, in dodecylphosphocholine micelles, we obtained excellent 500 and 800 MHz NMR spectra, suggesting that the peptide has a preference for a zwitterionic membrane-like environment. The final NMR structures demonstrated a well-defined helical peptide with a backbone rmsd of 0.47 +/- 0.18 A. Four of the five tryptophan residues, as well as the tyrosine residue, formed a "collar" of aromatic residues along the axial length of the helix. By analogy to related tryptophan-rich antimicrobial peptides, the structure indicates that the aromatic residues of the HIV peptide are positioned within the membrane-water interface of a phospholipid bilayer. This is confirmed by the observation of direct NOEs between the aromatic residues of the peptide to the headgroup and interfacial protons of prototonated dodecylphosphocholine. The bulk of the polar residues are positioned on one face of this structure, with the hydrophobic phenylalanine side chain on the opposing face, forming an amphipathic structure. This work shows that the Trp-rich membrane-proximal region of HIV and related viruses can bind to the surfaces of zwitterioninc membranes in a "Velcro-like" manner.     

Structural analysis of synthetic peptide fragments from EmrE,a multidrug resistance protein,in a membrane-mimetic environment

EmrE, a multidrug resistance protein from Escherichia coli, renders the bacterium resistant to a variety of cytotoxic drugs by active translocation out of the cell. The 110-residue sequence of EmrE limits the number of structural possibilities that can be envisioned for this membrane protein. Four helix bundle models have been considered [Yerushalmi, H., Lebendiker, M., and Schuldiner, S. (1996) J. Biol. Chem. 271, 31044-31048]. The validity of EmrE structural models has been probed experimentally by investigations on overlapping peptides (ranging in length from 19 to 27 residues), derived from the sequence of EmrE. The choice of peptides was made to provide sequences of two complete, predicted transmembrane helices (peptides H1 and H3) and two helix-loop-helix motifs (peptides A and B). Peptide (B) also corresponds to a putative hairpin in a speculative beta-barrel model, with the "Pro-Thr-Gly" segment forming a turn. Structure determination in SDS micelles using NMR indicates peptide H1 to be predominantly helical, with helix boundaries in the micellar environment corroborating predicted helical limits. Peptide A adopts a helix-loop-helix structure in SDS micelles, and peptide B was also largely helical in micellar environments. An analogue peptide, C, in which the central "Pro-Thr-Gly" was replaced by "(D)Pro-Gly" displays local turn conformation at the (D)Pro-Gly segment, but neither a continuous helical stretch nor beta-hairpin formation was observed. This study implies that the constraints of membrane and micellar environments largely direct the structure of transmembrane peptides and proteins and study of judiciously selected peptide fragments can prove useful in the structural elucidation of membrane proteins.     

Solution structure of the fifth and sixth transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the fifth and sixth transmembrane segments of the bovine mitochondrial oxoglutarate carrier (OGC) and of the hydrophilic loop that connects them were studied by CD and NMR spectroscopies. Peptides F215-R246, W279-K305 and P257-L278 were synthesized and structurally characterized. CD data showed that at high concentrations of TFE and SDS all peptides assume α-helical structures. ¹H-NMR spectra of the three peptides in TFE/water were fully assigned and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides were generated by distance geometry calculations. A well-defined α–helix was found in the region L220-V243 of peptide F215-R246 (TMS-V), in the region P284-M303 of peptide W279-K305 (TMS-VI) and in the region N261-F275 of peptide P257-L278 (hydrophilic loop). The helix L220-V243 exhibited a sharp kink at P239, while a little bend around P291 was observed in the helical region P284-M303. Fluorescence studies performed on peptide W279-K305, alone and together with other transmembrane segments of OGC, showed that the W279 fluorescence was quenched upon addition of peptide F215-R246, but not of peptides K21-K46, R78-R108 and P117-A149 suggesting a specific interaction between TMS-V and TMS-VI of OGC.     

Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662–683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning.     

Coarse-grained molecular dynamics simulations of membrane proteins and peptides

Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.     

Functional signal peptide reduces bilayer thickness of phosphatidylcholine liposomes.

To investigate the interaction between a signal peptide and the lipid bilayer, two kinds of peptides, L8-M5 (L8 = MRL8PLAALG, M5 = KVFER) and L14-M5 (L14 = MRL14PLAALG), were examined in membranes composed of dioleoylphosphatidylcholine (DOPC). Peptides L8 and L14 are artificially designed signal sequences, and M5 is the N-terminal five residues of human lysozyme; L8 mediated effective secretion of human lysozyme in yeast, while L14 did not [Yamamoto, Y., et al. (1987) Biochem. Biophys. Res. Commun. 149, 431-436]. DOPC liposomes incorporating L8-M5 or L14-M5 were observed by electron cryomicroscopy as pairs of concentric circles, and the separation of the bilayer was measured along the membrane. Peptide L8-M5 was found to reduce the bilayer thickness, but L14-M5 did not. CD measurements revealed that L8-M5 adopted an alpha-helical conformation with random coil in the liposome membranes and that L14-M5 adopted a more helical and less random conformation than L8-M5. Fluorescence spectroscopy using both aqueous and membranous probes revealed that L8-M5 destabilized the lipid bilayer more strongly than L14-M5. These results suggest that functional L8-M5 reduces the bilayer thickness and destabilizes the lipid bilayer and that these activities are important for signal peptide function.     

Computationally designed peptide inhibitors of protein-protein interactions in membranes

We recently reported a computational method (CHAMP) for designing sequence-specific peptides that bind to the membrane-embedded portions of transmembrane proteins. We successfully applied this method to design membrane-spanning peptides targeting the transmembrane domains of the alpha IIb subunit of integrin alpha IIbbeta 3. Previously, we demonstrated that these CHAMP peptides bind specifically with reasonable affinity to isolated transmembrane helices of the targeted transmembrane region. These peptides also induced integrin alpha IIbbeta 3 activation due to disruption of the helix-helix interactions between the transmembrane domains of the alpha IIb and beta 3 subunits. In this paper, we show the direct interaction of the designed anti-alpha IIb CHAMP peptide with isolated full-length integrin alpha IIbbeta 3 in detergent micelles. Further, the behavior of the designed peptides in phospholipid bilayers is essentially identical to their behavior in detergent micelles. In particular, the peptides assume a membrane-spanning alpha-helical conformation that does not disrupt bilayer integrity. The activity and selectivity of the CHAMP peptides were further explored in platelets, comfirming that anti-alpha IIb activates wild-type alpha IIbbeta 3 in whole cells as a result of its disruption of the protein-protein interactions between the alpha and beta subunits in the transmembrane regions. These results demonstrate that CHAMP is a successful chemical biology approach that can provide specific tools for probing the transmembrane domains of proteins.     

Structural characterization of the transmembrane domain from subunit e of yeast F1Fo-ATP synthase: a helical GXXXG motif located just under the micelle surface

F1Fo-ATP synthase is a large multiprotein complex, including at least 10 subunits in the membrane-bound Fo-sector. One of these Fo proteins is subunit e (Su e), involved in the stable dimerization of F1Fo-ATP synthase, and required for the establishment of normal cristae membrane architecture. As a step toward enabling structure-function studies of the Fo-sector, the Su e transmembrane region was structurally characterized in micelles. Based on a series of NMR and CD (circular dichroism) studies, a structural model of the Su e/micelle complex was constructed, indicating Su e is largely helical, and emerges from the micelle with Arg20 near the phosphate head groups. Su e only adopts this folded conformation in the context of the micelle, and is essentially disordered in DMSO, water or trifluoroethanol/water. Within the micelle the C-terminal Ala10-Arg20 stretch is helical, while the region N-terminal may be transiently helical, based on negative CSI (chemical shift index) values. The Ala10-Arg20 helix contains the G14XXXG18 motif, which has been proposed to play an important role in dimer formation with another protein from the Fo-sector. The Gly on the C-terminal end of this motif (Gly18) is slightly more mobile than the more buried Gly14, based on NMR order parameter measurements (Gly14 S2 = 0.950; Gly18 S2 = 0.895). Only one Su e transmembrane peptide is bound per micelle, and micelles are 22-23 A in diameter, composed of 51 +/- 4 dodecylphosphocholine detergent molecules. Although there is no evidence for Su e homodimerization via the transmembrane domain, potentially synergistic roles for N-terminal (membrane) and C-terminal (soluble) domain interactions may still occur. Furthermore, the presence of a buried charged residue (Arg7) suggests there may be interactions with other Fo-sector protein(s) that stabilize this charge, and possibly drive the folding of the N-terminal 9 residues of the transmembrane domain.     

A solvent model for simulations of peptides in bilayers. II. Membrane-spanning alpha-helices

We describe application of the implicit solvation model (see the first paper of this series), to Monte Carlo simulations of several peptides in bilayer- and water-mimetic environments, and in vacuum. The membrane-bound peptides chosen were transmembrane segments A and B of bacteriorhodopsin, the hydrophobic segment of surfactant lipoprotein, and magainin2. Their conformations in membrane-like media are known from the experiments. Also, molecular dynamics study of surfactant lipoprotein with different explicit solvents has been reported (Kovacs, H., A. E. Mark, J. Johansson, and W. F. van Gunsteren. 1995. J. Mol. Biol. 247:808-822). The principal goal of this work is to compare the results obtained in the framework of our solvation model with available experimental and computational data. The findings could be summarized as follows: 1) structural and energetic properties of studied molecules strongly depend on the solvent; membrane-mimetic media significantly promote formation of alpha-helices capable of traversing the bilayer, whereas a polar environment destabilizes alpha-helical conformation via reduction of solvent-exposed surface area and packing; 2) the structures calculated in a membrane-like environment agree with the experimental ones; 3) noticeable differences in conformation of surfactant lipoprotein assessed via Monte Carlo simulation with implicit solvent (this work) and molecular dynamics in explicit solvent were observed; 4) in vacuo simulations do not correctly reproduce protein-membrane interactions, and hence should be avoided in modeling membrane proteins.     

SMAP-29 has two LPS-binding sites and a central hinge.

The CD spectra of SMAP-29, an antimicrobial peptide from sheep, showed disordered structure in aqueous buffers, and significant helicity in membrane-like environments, including SDS micelles, lipopolysaccharide (LPS) dispersions, and trifluoroethanol buffer systems. A structure determined by NMR in 40% perdeuterated trifluoroethanol indicated that residues 8-17 were helical, residues 18-19 formed a hinge, and residues 20-28 formed an ordered, hydrophobic segment. SMAP-29 was flexible in 40% trifluoroethanol, forming two sets of conformers that differed in the relative orientation of the N-terminal domain. We used a chromogenic Limulus assay to determine the EC50 of the peptide (the concentration that bound 50% of the added LPS). Studies with full-length and truncated SMAP-29 molecules revealed that each end of the holopeptide contained an LPS-binding domain. The higher affinity LPS-binding domain was situated in the flexible N-terminal portion. LPS binding to full-length SMAP-29 showed positive cooperativity, so the EC50 of the peptide (2.6 microm) was considerably lower than that of the individual LPS-binding domains. LPS-binding studies with a mixture of truncated peptides revealed that this cooperativity was primarily intramolecular (i.e. involving the N- and C-terminal LPS-binding sites of the same peptide molecule). CAP-18[106 -142], an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N- and C-terminal LPS-binding domains, had an EC50 of 2.5 microm, and bound LPS with positive cooperativity. We conclude that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity.