Electron paramagnetic resonance studies of an integral membrane peptide inserted into aligned phospholipid bilayer nanotube arrays

This communication reports for the first time the determination of the helical tilt of an integral membrane peptide inserted into aligned phospholipids bilayer nanotube arrays using spin label EPR spectroscopy. Also, we demonstrate herein how the helical tilt of the peptide can be easily calculated using the hyperfine splitting values gleaned from a perpendicularly aligned bilayer in phospholipid bilayer nanotube arrays. EPR spectral simulations were used to verify the method.     

Magnetically aligned phospholipid bilayers with large q ratios stabilize magnetic alignment with high order in the gel and L(alpha) phases

The magnetic alignment behavior ofbicelles (magnetically alignable phospholipid bilayered membranes) as a function of the q ratio (1,2-dihexanoyl-sn-glycerol phosphatidylcholine/1,2-dimyristoyl-sn-glycerol phosphatidylcholine mole ratio) and temperature was studied by spin-labeled X-band electron paramagnetic resonance (EPR) spectroscopy and solid-state 2H and 31P NMR spectroscopy. Well-aligned bicelle samples were obtained at 45 degrees C for q ratios between 2.5 and 9.5 in both the EPR and NMR spectroscopic studies. The molecular order of the system, S(mol), increased as the q ratio increased and as the temperature decreased. For higher q ratios (> or = 5.5), bicelles maintained magnetic alignment when cooled below the main phase transition temperature (approximately 30 degrees C when in the presence of lanthanide cations), which is the first time, to our knowledge, that bicelles were magnetically aligned in the gel phase. For the 9.5 q ratio sample at 25 degrees C, S(mol) was calculated to be 0.83 (from 2H NMR spectra, utilizing the isotopic label perdeuterated 1,2-dimyristoyl-sn-glycerol phosphatidylcholine) and 0.911 (from EPR spectra utilizing the spin probe 3beta-doxyl-5alpha-cholestane). The molecular ordering of the high q ratio bicelles is comparable to literature values of S(mol) for both multilamellar vesicles and macroscopically aligned phospholipid bilayers on glass plates. The order parameter S(bicelle) revealed that the greatest degree of bicelle alignment was found at higher temperatures and larger q ratios (S(bicelle) = -0.92 for q ratio 8.5 at 50 degrees C).     

Solid-state 2H NMR studies of the effects of cholesterol on the acyl chain dynamics of magnetically aligned phospholipid bilayers

We report the utilization of magnetically aligned phospholipid bilayers (bicelles) to study the effects of cholesterol in phospholipid bilayers for both chain perdeuterated DMPC and partially deuterated alpha-[2,2,3,4,4,6-d(6)]-cholesterol using (2)H solid-state NMR spectroscopy. The quadrupolar splittings at 40 degrees C were 25.5 and 37.7 kHz, respectively, for the 2,4-(2)H(eq) and 2,4-(2)H(ax) deuterons when the bilayer normal of the discs was aligned perpendicular to the static magnetic field. The quadrupolar splittings were doubled when Yb(3+) ions were added to flip the bicelles 90 degrees such that the bilayer normal was colinear with the magnetic field. The results suggest that cholesterol is incorporated into the bicelle discs. For chain perdeuterated DMPC-d(54), incorporated into DMPC-DHPC bicelle discs, the individual quadrupolar splittings of the methylene and methyl groups doubled on going from the perpendicular to the parallel alignment. Also, the presence of cholesterol increased the overall ordering of the acyl chains of the phospholipids. S(CD) (i) calculations were extracted directly from the (2)H quadrupolar splittings of the chain perdeuterated DMPC. The order parameter, S(CD) (i), calculations clearly indicated an overall degree of ordering of the acyl chains in the presence of cholesterol. We also noted a disordering effect at higher temperatures. This study demonstrates the ease with which (2)H order parameters can be calculated utilizing magnetically aligned phospholipid bilayers when compared with randomly dispersed membrane samples.     

NMR methods for studying the structure and dynamics of oncogenic and antihistaminic peptides in biomembranes

We present several applications of both wide-line and magic angle spinning (MAS) solid-state NMR of bicelles in which are embedded fragments of a tyrosine kinase receptor or enkephalins. The magnetically orientable bicelle membranes are shown to be of particular interest for studying the functional properties of lipids and proteins in a state that is very close to their natural environment. Quadrupolar, dipolar and chemical shielding interactions can be used to determine minute alterations of internal membrane dynamics and the orientation of peptides with respect to the membrane plane. MAS of bicelles can in turn lead to high-resolution proton spectra of hydrated membranes. Using deuterium-proton contrast methods one can then obtain pseudo-high-resolution proton spectra of peptides or proteins embedded in deuterated membranes and determine their atomic 3D structure using quasi-conventional liquid-state NMR methods.     

Magnetic resonance investigations of lipid motion in isotropic bicelles

The dynamics of DMPC in different isotropic bicelles have been investigated by NMR and EPR methods. The local dynamics were obtained by interpretation of 13C NMR relaxation measurements of DMPC in the bicelles, and these results were compared to EPR spectra of spin-labeled lipids. The overall size of the bicelles was investigated by PFG NMR translational diffusion measurements. The dynamics and relative sizes were compared among three different bicelles: [DMPC]/[DHPC] = 0.25, [DMPC]/[DHPC] = 0.5, and [DMPC]/[CHAPS] = 0.5. The local motion is found to depend much more strongly on the choice of the detergent, rather than the overall size of the bicelle. The results provide an explanation for differences in apparent dynamics for different peptides, which are bound to bicelles. This in turn determines under what conditions reasonable NMR spectra can be observed. A model is presented in which extensive local motion, in conjunction with the overall size, affects the spectral properties. An analytical expression for the size dependence of the bicelles, relating the radius of the bilayer region with physical properties of the detergent and the lipid, is also presented.     

Comparing the structural topology of integral and peripheral membrane proteins utilizing electron paramagnetic resonance spectroscopy

The alignment of membrane proteins provides pertinent structural and dynamic information. Structural topology data gleaned from such studies can be used to determine the functional mechanisms associated with a wide variety of integral membrane proteins. In this communication, we successfully demonstrate, for the first time, the determination of the structural topology and helical tilt of an antimicrobial peptide magainin 2 using aligned X-band spin-label EPR spectroscopic techniques. This novel comparison unlocks many possibilities utilizing EPR spectroscopy to probe antimicrobial peptide topologies with increased sensitivity and may also give further clues to elucidate their corresponding mechanisms.     

Evidence for effect of GM1 on opioid peptide conformation: NMR study on leucine enkephalin in ganglioside-containing isotropic phospholipid bicelles

Enkephalins are endogenous neuropeptides that have opioid-like activities and compete with morphines for the receptor binding. The binding of these neuropeptides to membrane appears crucial since enkephalins interact with the nerve cell membranes to achieve bioactive conformations that fit onto multiple receptor sites (micro, delta, and kappa). Using NMR spectroscopy, we have determined the solution structure of the small opiate pentapeptide leucine enkephalin in the presence of isotropic phospholipid bicelles: phosphocholine bicelles (DMPC:CHAPS 1:4) and phosphocholine bicelles doped with ganglioside GM1 (DMPC:CHAPS:GM1 1:4:0.3). Bicelles containing GM1 were found to interact strongly with leucine enkephalin, whereas a somewhat weaker interaction was observed in the case of bicelles without GM1. Structure calculation from torsion angles, chemical shifts, and NOE-based distance constraints explored that the peptide could flexibly switch between several mu- and delta-selective conformations in both the bicelles though micro-selective conformations turned out to be geometrically preferred in each bicellar system. A detailed analysis of the structures presented supports the variance over the singly associated conformation of enkephalin in nerve cell membranes.     

Synthesis and Properties of Isoindoline Nitroxide‐containing Porphyrins

Isoindoline nitroxide‐containing porphyrins were synthesized by the reaction of 5‐phenyldipyrromethane and 5‐(4′‐nitrophenyl)‐dipyrromethane with 5‐formyl‐1,1,3,3‐tetramethylisoindolin‐2‐yloxyl using the Lindsey method. These spin‐labeled porphyrins were further characterized by MS, UV, FTIR, ¹H‐NMR, cyclic voltammetry, electron paramagnetic resonance (EPR), and fluorescence spectroscopy. The electrochemical assay demonstrated that these isoindoline nitroxides‐containing porphyrins had similar electrochemical and redox properties as 5‐carboxy‐1,1,3,3‐tetramethylisoindolin‐2‐yloxyl. Electron paramagnetic resonance test exhibited these porphyrins possessed the hyperfine splittings and characteristic spectra of isoindoline nitroxides, with typical nitroxide g‐values and nitrogen isotropic hyperfine coupling constants. Fluorescence spectroscopy revealed that these porphyrins indicated fluorescence suppression characteristic of nitroxide–fluorophore systems. Moreover, their reduced isoindoline nitroxide‐containing porphyrins eliminated the fluorescence suppression and displayed strong fluorescence. Thus, these isoindoline nitroxide‐containing porphyrins may be considered as the potential fluorescent and EPR probes.     

Determining the orientation and localization of membrane-bound peptides

Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance.     

High-resolution NMR spectroscopy of membrane proteins in aligned bicelles

High-resolution solid-state NMR spectra can be obtained from uniformly (15)N-labeled membrane proteins in magnetically aligned bicelles. Fast uniaxial diffusion about the axis of the bilayer normal results in single-line spectra that contain the orientational information necessary for protein structure determination.     

Radical trapping properties of 3-aryl-2H-benzo[1,4]oxazin-4-oxides

A series of 3-aryl-2H-benzo[1,4]oxazin-4-oxides was prepared, and their ability to trap free radicals was investigated by EPR spectroscopy. In organic solvents, these compounds were able to efficiently scavenge all carbon- and oxygen-centered radicals tested, giving very persistent aminoxyls, except with superoxide anion whose spin adducts were unstable. The main feature of these nitrones as spin traps lies in the possibility to recognize the initial radical trapped. In fact, besides a g-factor and aminoxyl nitrogen EPR coupling constant dependence on the species trapped, the EPR spectra also show different patterns due to hyperfine splittings characteristic of the radical scavenged. This last important feature was investigated by means of density functional theory calculations.     

Distance measurements between paramagnetic centers and a planar object by matrix Mims electron nuclear double resonance

Frequency-domain electron nuclear double resonance (ENDOR), two time-domain electron nuclear double resonance techniques, and electron spin echo envelope modulation spectroscopy are compared with respect to their merit in measurements of small hyperfine couplings to nuclei with intermediate gyromagnetic ratio such as 31P. The frequency-domain Mims ENDOR experiment is found to provide the most faithful line shapes. In the limit of long electron-nuclear distances of more than 0.5 nm, sensitivity of this experiment is optimized by matching the first interpulse delay to the transverse relaxation time of the electron spins. In the same limit, Mims ENDOR efficiency scales inversely with the sixth power of distance. Hyperfine splittings as small as 33 kHz can be detected, corresponding to an electron-31P distance of 1 nm. In systems, where a certain kind of nuclei is distributed in a plane, measurements of intermolecular hyperfine couplings can be analyzed in terms of a distance of closest approach of a paramagnetic center to that plane. By applying this technique to spin-labeled lipids in a fully hydrated lipid bilayer it is found that for a fraction of lipids, chain tilt angles can be 25 degrees larger than the mean tilt angle of the lipid chains. This model of all-trans hydrocarbon chains with a broad distribution of tilt angles is also consistent with orientation selection effects in high-field ENDOR spectra.     

Simultispin: A versatile graphical user interface for the simulation of solid-state continuous wave EPR spectra

Solid-state continuous wave (cw) electronic paramagnetic resonance (EPR) spectroscopy is particularly suitable for metal complex analysis. Extracting magnetic parameters by simulation is often necessary to describe the electronic structure of the studied molecular compounds that can have various electronic spin states and characterized by different parameters like g-values, hyperfine coupling or zero field splitting values. Easyspin toolbox on MATLAB is a powerful tool, but for the user, it requires spending time with coding and could discourage nonexperts. Facing this context, we have developed a graphical user interface called Simultispin, dedicated to solid-state cw-EPR spectra simulation. Some examples of experimental spectra of metal complexes (mixture of low spin and high spin Fe^III complexes, dynamic disorder of a Cu^II complex, photogeneration of a Mn^III complex), highlighting specific solid-state functions, are described and analyzed based on simulations performed with Simultispin. We hope that its ergonomy and the ease to set up a complete set of parameters to get reliable simulations could help a large EPR community to improve the efficiency of their interpretations.     

Lipid-ethanol interaction studied by NMR on bicelles

The interaction of ethanol with phospholipids was studied in bicelles at a physiologically relevant ethanol concentration of 20 mM and a lipid content of 14 wt % by high-resolution NMR. Transient association of ethanol with magnetically aligned bicelles imparts a small degree of anisotropy to the solute. This anisotropy allows detection of residual (1)H-(1)H and (1)H-(13)C dipolar couplings, which are superimposed on scalar couplings. Residual (2)H NMR quadrupole splittings of isotope-labeled ethanol were measured as well. The analysis of residual tensorial interactions yielded information on the orientation and motions of ethanol in the membrane-bound state. The fraction of phosphatidylcholine-bound ethanol was determined independently by gas chromatography and NMR. About 4% of ethanol is bound to phosphatidylcholine at a bicelle concentration of 14 wt % at 40 degrees C. Free and bound ethanol are in rapid exchange. The lifetime of ethanol association with phosphatidylcholine membranes is of the order of a few nanoseconds.     

Solid-state NMR spectroscopic studies of an integral membrane protein inserted into aligned phospholipid bilayer nanotube arrays

This communication demonstrates for the first time that solid-state NMR spectroscopic studies can be used to investigate aligned phospholipid bilayer nanotube arrays. Also, an integral membrane peptide can be successfully incorporated into the oriented phospholipid bilayer nanotube arrays and studied with 2H solid-state NMR spectroscopy.     

Positional and angular dependence of hyperfine interactions in cyclic and bicyclic iminoxy radicals with CO or CH2 group – DFT and EPR studies

UB1LYP method was used to study the geometries together with hyperfine coupling constants (hfccs) and natural atomic occupancies (NAO) for the cyclic (cyclohexanone-type) and bicyclic (camphor-type) iminoxy radicals. The positional and angular dependence of the hyperfine interactions, affected by radical substituents and conformations, was analyzed in terms of different mechanisms of spin density transmission. The calculations predicted a significant distortion of regular conformations and change of hyperfine couplings upon introduction of CO into cyclohexane iminoxyl and the CNO spin label into a boat cyclohexane. Hyperfine splittings of the EPR spectra were compared with the computed hfccs to verify their assignment.     

On the orientation of a designed transmembrane peptide: toward the right tilt angle?

The orientation of the transmembrane peptide WALP23 under small hydrophobic mismatch has been assessed through long-time-scale molecular dynamics simulations of hundreds of nanoseconds. Each simulation gives systematically large tilt angles (>30 degrees). In addition, the peptide visits various azimuthal rotations that mostly depend on the initial conditions and converge very slowly. In contrast, small tilt angles as well as a well-defined azimuthal rotation were suggested by recent solid-state 2H NMR studies on the same system. To optimally compare our simulations with NMR data, we concatenated the different trajectories in order to increase the sampling. The agreement with 2H NMR quadrupolar splittings is spectacularly better when these latter are back-calculated from the concatenated trajectory than from any individual simulation. From these ensembled-average quadrupolar splittings, we then applied the GALA method as described by Strandberg et al. (Biophys J. 2004, 86, 3709-3721), which basically derives the peptide orientation (tilt and azimuth) from the splittings. We find small tilt angles (6.5 degrees), whereas the real observed tilt in the concatenated trajectory presents a higher value (33.5 degrees). We thus propose that the small tilt angles estimated by the GALA method are the result of averaging effects, provided that the peptide visits many states of different azimuthal rotations. We discuss how to improve the method and suggest some other experiments to confirm this hypothesis. This work also highlights the need to run several and rather long trajectories in order to predict the peptide orientation from computer simulations.     

Tailoring 13C labeling for triple-resonance solid-state NMR experiments on aligned samples of proteins

In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different (13)C labeling schemes with the purpose of overcoming the interfering effects of (13)C-(13)C dipole-dipole couplings in stationary samples. The membrane-bound form of the major coat protein of the filamentous bacteriophage Pf1 was used as an example of a well-characterized helical membrane protein. Aligned protein samples randomly enriched to 35% (13)C in all sites and metabolically labeled from bacterial growth on media containing [2-(13)C]-glycerol or [1,3-(13)C]-glycerol enables direct (13)C detection in solid-state NMR experiments without the need for homonuclear (13)C-(13)C dipole-dipole decoupling. The (13)C-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent (15)N-detected spectra. The isotopic labeling pattern was analyzed for [2-(13)C]-glycerol and [1,3-(13)C]-glycerol as metabolic precursors by solution-state NMR of micelle samples. Polarization inversion spin exchange at the magic angle (PISEMA) and other solid-state NMR experiments work well on 35% random fractionally and metabolically tailored (13)C-labeled samples, in contrast to their failure with conventional 100% uniformly (13)C-labeled samples.     

Membrane alignment of the pore-forming component TatA(d) of the twin-arginine translocase from Bacillus subtilis resolved by solid-state NMR spectroscopy

The twin-arginine translocase (Tat) provides protein export in bacteria and plant chloroplasts and is capable of transporting fully folded proteins across the membrane. We resolved the conformation and membrane alignment of the pore-forming subunit TatA(d) from Bacillus subtilis using solid-state NMR spectroscopy. The relevant structured part of the protein, TatA(2-45), contains a transmembrane segment (TMS) and an amphiphilic helix (APH). It was reconstituted in planar bicelles, which represent the lipid environment of a bacterial membrane. The SAMMY solid-state NMR experiment was used to correlate (15)N chemical shifts and (1)H-(15)N dipolar couplings in the backbone and side chains of the (15)N-labeled protein. The observed wheel-like patterns ("PISA wheels") in the resulting 2-dimensional spectra confirm the α-helical character of the two segments and reveal their alignment in the lipid bilayer. Helix tilt angles (τ(TMS) = 13°, τ(APH) = 64°) were obtained from uniformly labeled protein, and azimuthal rotations (ρ(Val15) = 235°, ρ(Ile29) = 25°) were obtained from selective labels. These constraints define two distinct families of allowed structures for TatA in the membrane-bound state. The manifold of solutions could be narrowed down to a unique structure by using input from a liquid-state NMR study of TatA in detergent micelles, as recently described [Hu, Y.; Zhao, E.; Li, H.; Xia, B.; Jin, C. J. Am. Chem. Soc. 2010, DOI: 10.1021/ja1053785]. Interestingly, the APH showed an unexpectedly slanted alignment in the protein, different from that of the isolated APH peptide. This finding implies that the amphiphilic region of TatA is not just a flexible attachment to the transmembrane anchor but might be able to form intra- or even intermolecular salt-bridges, which could play a key role in pore assembly.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label

X-band electron paramagnetic resonance (EPR) spectroscopy was used to study the structural and dynamic properties of magnetically aligned phospholipid bilayers utilizing a variety of phosphocholine spin labels (PCSL) as a function oftemperature. 1-Palmitoyl-2-[n-(4,4-dimethyloxazolidine-N-oxyl)stearoyl]-sn-glycero-3-phosphocholine (n-PCSL) in which a nitroxide group was attached to the different acyl chain positions of the phospholipid (n = 5, 7, 12, and 14) were used as an EPR spin probe to investigate magnetically aligned phospholipid bilayers from the plateau (near to the headgroup) region to the end of the acyl chain (center of the bilayers). The addition of certain types of paramagnetic lanthanide ions changes the overall magnetic susceptibility anisotropy tensor of the bicelles, such that the bicelles flip with their bilayer normal either parallel or perpendicular to the magnetic field. The present study reveals for the first time that, in the case of the n-PCSL, the bilayer normal is aligned parallel and perpendicular to the magnetic field in the presence of lanthanide ions having positive delta(chi) (e.g., Tm3+) and negative delta(chi) (e.g., Dy3+), respectively. The magnetic alignment of the bilayers and the corresponding segmental molecular order parameter, S(mol), were investigated as a function of the temperature. The S(mol) values decrease in the following order, 5-PCSL > 7-PCSL > 12-PCSL > 14-PCSL, for the magnetically aligned phospholipid bilayers. Also, the variable temperature study indicates that, by increasing the temperature, the order parameters S(mol) decreased for all the n-PCSLs. The results indicate that magnetically aligned phospholipid bilayers represent an excellent model membrane system for X-band EPR studies.