Alamethicin in bicelles: Orientation,aggregation, and bilayer modification as a function of peptide concentration

Alamethicin is a 19-amino-acid residue hydrophobic peptide of the peptaibol family that has been the object of numerous studies for its ability to produce voltage-dependent ion channels in membranes. In this work, for the first time electron paramagnetic resonance spectroscopy was applied to study the interaction of alamethicin with oriented bicelles. We highlighted the effects of increasing peptide concentrations on both the peptide and the membrane in identical conditions, by adopting a twofold spin labeling approach, placing a nitroxide moiety either on the peptide or on the phospholipids. The employment of bicelles affords additional spectral resolution, thanks to the formation of a macroscopically oriented phase that allows to gain information on alamethicin orientation and dynamics. Moreover, the high viscosity of the bicellar solution permits the investigation of the peptide aggregation properties at physiological temperature. We observed that, at 35 °C, alamethicin adopts a transmembrane orientation with the peptide axis forming an average angle of 25° with respect to the bilayer normal. Moreover, alamethicin maintains its dynamics and helical tilt constant at all concentrations studied. On the other hand, by increasing the peptide concentration, the bilayer experiences an exponential decrease of the order parameter, but does not undergo micellization, even at the highest peptide to lipid ratio studied (1:20). Finally, the aggregation of the peptide at physiological temperature shows that the peptide is monomeric at peptide to lipid ratios lower than 1:50, then it aggregates with a rather broad distribution in the number of peptides (from 6 to 8) per oligomer.     

Diffusion and dynamics of penetratin in different membrane mimicking media

The interaction between the cell-penetrating peptide (CPP) penetratin and different membrane mimetic environments has been investigated by two different NMR methods: ¹⁵N spin relaxation and translational diffusion. Diffusion coefficients were measured for penetratin in neutral and in negatively charged bicelles of different size, in sodium dodecyl sulfate micelles (SDS), and in aqueous solution. The diffusion coefficients were used to estimate the amount of free and bicelle/micelle-bound penetratin and the results revealed that penetratin binds almost fully to all studied membrane mimetics. ¹⁵N relaxation data for three sites in penetratin were interpreted with the model-free approach to obtain overall and local dynamics. Overall correlation times for penetratin were in agreement with findings for other peptides of similar size in the same solvents. Large differences in order parameters were observed for penetratin in the different membrane mimetics. Negatively charged surfaces were seen to restrict motional flexibility, while a more neutral membrane mimetic did not. This indicates that although the peptide binds to both bicelles and SDS micelles, the interaction between penetratin and the various membrane mimetics is different.     

Structural Basis for the Association of the Redox-sensitive Target of Rapamycin FATC Domain with Membrane-mimetic Micelles

The target of rapamycin (TOR) is a conserved eukaryotic Ser/Thr kinase that regulates cellular growth in response to the nutrient and energy state. TOR signaling plays an important role in the development of diseases such as cancer, obesity, and diabetes and in different redox-sensitive processes (hypoxia, apoptosis, and aging). Because TOR has been detected at different cellular membranes and in the nucleus, its localization may influence the specific signaling readout. To better understand how TOR can associate with different membranes, the lipid-binding properties of the redox-sensitive yeast TOR1 FATC domain (y1fatc) have been characterized by solution NMR spectroscopy. Binding studies with different lipids indicate that y1fatc interacts specifically with a membrane-mimetic environment but appears not to recognize a specific lipid headgroup. In both, the structures of oxidized and reduced micelle-bound y1fatc, residues Ile-2456 to Trp-2470 of the lipid-binding motif form a hydrophobic bulb that has a rim of charged residues. The diffusion constants for both micelle-bound states are consistent with the rotational correlation times from the analysis of the ¹⁵N relaxation data. Based on the K_d values, the oxidized form (K_d ∼ 0.31 mm) binds dodecyl phosphocholine micelles slightly tighter than the reduced form (K_d ∼ 1.86 mm). Binding studies with y1fatc in which one or both tryptophans (Trp-2466 and Trp-2470) were replaced by alanine suggest that these residues are important for the exact positioning in the membrane and that the other aromatic (His-2462, Tyr-2463, and Phe-2469) and aliphatic residues (Ile-2456, Leu-2459, Ile-2464, and Pro-2468) in the lipid-binding motif contribute significantly to the affinity.     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.     

Investigating the structural and dynamic properties of n-doxylstearic acid in magnetically-aligned phospholipid bilayers by X-band EPR spectroscopy

X-band electron paramagnetic resonance (EPR) spectroscopy has been employed to investigate the dynamic properties of magnetically-aligned phospholipid bilayers (bicelles) based on the molecular order parameters (S(mol)), the hyperfine splitting values and the line shapes of the EPR spectra. For the first time, a series of EPR spectra of n-doxylstearic acid spin-labels (n = 5, 7, 12, and 16) incorporated into Tm3+-doped parallel-aligned, Dy3+-doped perpendicular-aligned, and randomly dispersed 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelles with respect to the direction of the static magnetic field have been investigated as a function of cholesterol content and temperature variation to characterize the orientational aspects along the hydrocarbon acyl chains. Important general observations are that under conditions for which the bicelle is poised in the liquid crystalline phase, the degree of ordering decreases as the nitroxide moiety is transferred toward the end of the stearic acid acyl chains. The addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC phospholipid bilayers and increases the chain order. However, increasing the temperature of the bicelle system decreases the chain order. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other biological and model membrane systems. The results indicate that magnetically-aligned phospholipid bilayers are an excellent model membrane system.     

Reconstitution and alignment by a magnetic field of a β-barrel membrane protein in bicelles

A protocol is described for the reconstitution of a transmembrane β-barrel protein domain, tOmpA, into lipid bicelles. tOmpA is the largest protein to be reconstituted in bicelles to date. Its insertion does not prevent bicelles from orienting with their plane either parallel or perpendicular to the magnetic field, depending on the absence or presence of paramagnetic ions. In the latter case, tOmpA is shown to align with the axis of the β-barrel parallel to the magnetic field, i.e. perpendicular to the plane of the bilayer, an orientation conforming to that in natural membranes and favourable to structural studies by solid-state NMR. Reconstitution into bicelles may offer an interesting approach for structural studies of membrane proteins in a medium resembling a biological membrane, using either NMR or other biophysical techniques. Our data suggest that alignment in the magnetic field of membrane proteins included into bicelles may be facilitated if the protein is folded as a β-barrel structure.     

Solution NMR of membrane proteins in bilayer mimics: Small is beautiful, but sometimes bigger is better

Considerable progress has been made recently on solution NMR studies of multi-transmembrane helix membrane protein systems of increasing size. Careful correlation of structure with function has validated the physiological relevance of these studies in detergent micelles. However, larger micelle and bicelle systems are sometimes required to stabilize the active forms of dynamic membrane proteins, such as the bacterial small multidrug resistance transporters. Even in these systems with aggregate molecular weights well over 100 kDa, solution NMR structural studies are feasible—but challenging.     

The effects of temperature,salinity, concentration and PEGylated lipid on the spontaneous nanostructures of bicellar mixtures

The self-assembling morphologies of low-concentration (mostly 1 and 10 mg/mL) bicellar mixtures composed of zwitterionic dipalmitoyl (di-C₁₆) phosphatidylcholine (DPPC), dihexanoyl (di-C₆) phosphatidylcholine (DHPC), and negatively charged dipalmitoyl (di-C₁₆) phosphatidylglycerol (DPPG) were investigated using small angle neutron scattering, dynamic light scattering and transmission electron microscopy. A polyethylene glycol conjugated (PEGylated) lipid, distearoyl phosphoethanolamine-[methoxy (polyethyleneglycol)-2000] (PEG2000-DSPE), was incorporated in the system at 5 mol% of the total lipid composition. The effects of several parameters on the spontaneous structures were studied, including temperature, lipid concentration, salinity, and PEG2000-DSPE. In general, nanodiscs (bicelles) were observed at low temperatures (below the melting temperature, T_M of DPPC) depending on the salinity of the solutions. Nanodisc-to-vesicle transition was found upon the elevation of temperature (above T_M) in the cases of low lipid concentration in the absence of PEG2000-DSPE or high salinity. Both addition of PEG2000-DSPE and high lipid concentration stabilize the nanodiscs, preventing the formation of multilamellar vesicles, while high salinity promotes vesiculation and the formation of aggregation. This study suggests that the stability of such nanodiscs is presumably controlled by the electrostatic interactions, the steric effect induced by PEG2000-DSPE, and the amount of DHPC located at the disc rim.     

Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs

Nanodiscs, phospholipid bilayer assemblies of controlled size, were used to self-assemble bacteriorhodopsin (bR) into single trimers. Self-assembly at optimal bR to Nanodisc and phospholipid stoichiometry yielded particles containing three bR molecules. Analysis of solution small angle X-ray scattering indicated that bacteriorhodopsin is embedded in a discoidal phospholipid bilayer structure. Formation of trimers, as evidenced by visible circular dichroism of the retinal absorbance bands, is facilitated in Nanodiscs at a specific size threshold, suggesting that a critical bilayer area or amount of lipid is necessary to maintain a native oligomeric state. The lipid to bR ratio in the assembly process was also found to be an important factor in determining oligomerization state. These nanoscale bilayers offer the opportunity to understand and control the assembly of oligomeric integral membrane proteins critical to macromolecular recognition and cellular signaling.