Determination of Membrane Immersion Depth with O 2: A High-Pressure 19F NMR Study (original) (raw)
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The Journal of Physical Chemistry B, 2014
Paramagnetic relaxation enhancement (PRE) is a widely used approach for measuring long-range distance constraints in biomolecular solution NMR spectroscopy. In this paper, we show that 31 P PRE solid-state NMR spectroscopy can be utilized to determine the immersion depth of spinlabeled membrane peptides and proteins. Changes in the 31 P NMR PRE times coupled with modeling studies can be used to describe the spin-label position/amino acid within the lipid bilayer and the corresponding helical tilt. This method provides valuable insight on protein−lipid interactions and membrane protein structural topology. Solid-state 31 P NMR data on the 23 amino acid α-helical nicotinic acetylcholine receptor nAChR M2δ transmembrane domain model peptide followed predicted behavior of 31 P PRE rates of the phospholipid headgroup as the spin-label moves from the membrane surface toward the center of the membrane. Residue 11 showed the smallest changes in 31 P PRE (center of the membrane), while residue 22 shows the largest 31 P PRE change (near the membrane surface), when compared to the diamagnetic control M2δ sample. This PRE SS-NMR technique can be used as a molecular ruler to measure membrane immersion depth.
Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers
Journal of Magnetic Resonance, 2010
A new approach for determining the membrane immersion depth of a spin-labeled probe has been developed using paramagnetic relaxation enhancement (PRE) in solid-state NMR spectroscopy. A DOXYL spin label was placed at different sites of 1-palmitoyl-2-stearoyl-sn-glycero-3phosphocholine (PSPC) phospholipid bilayers as paramagnetic moieties and the resulting enhancements of the longitudinal relaxation (T 1 ) times of 31 P nuclei on the surface of the bilayers were measured by a standard inversion recovery pulse sequence. The 31 P NMR spin-lattice relaxation times decrease steadily as the DOXYL spin label moves closer to the surface as well as the concentration of the spin-labeled lipids increase. The enhanced relaxation vs. the position and concentration of spin-labels indicate that PRE induced by the DOXYL spin label are significant to determine longer distances over the whole range of the membrane depths. When these data were combined with estimated correlation times τ c , the r −6 -weighted, time-averaged distances between the spin labels and the 31 P nuclei on the membrane surface were estimated. The application of using this solid-state NMR PRE approach coupled with site-directed spin labeling (SDSL) may be a powerful method for measuring membrane protein immersion depth.
Biophysical Journal, 2003
The depth of insertion of an antimicrobial peptide, protegrin-1 (PG-1), in lipid bilayers is investigated using solidstate NMR. Paramagnetic Mn 21 ions bind to the surface of lipid bilayers and induce distance-dependent dipolar relaxation of nuclear spins. By comparing the signal dephasing of the peptide with that of the lipids, whose segmental depths of insertion are known, we determined the depths of several residues of PG-1 in 1,2 dilauryl-sn-glycero-3-phosphotidylcholine (DLPC) bilayers. We found that residues G2 at the N-terminus and F12 at the b-turn of the peptide reside near the membrane surface, whereas L5 and V16 are embedded in the acyl chain region. The depths increase in the order of G2 \ F12 \ L5 \ V16. These intensitydephasing results are confirmed by direct measurement of the paramagnetically enhanced 13 C transverse relaxation rates. The relative depths indicate that PG-1 is tilted from the bilayer normal, which is consistent with independent solid-state NMR measurements of PG-1 orientation in the same lipids (Yamaguchi et al., 2001). They also indicate that PG-1 is fully immersed in the lipid bilayer. However, a quantitative mismatch between the bilayer thickness and PG-1 length suggests a local thinning of the DLPC bilayer by 8-10 Å. The depth sensitivity of this Mn 21 dephasing technique is tunable with the Mn 21 concentration to focus on different regions of the lipid bilayer.
Journal of the American Chemical Society, 1998
We present a new solid-state NMR approach, based on 1 H spin diffusion with X-nucleus (15 N, 13 C, 31 P) detection, for investigating the structure of membrane proteins. For any segment with a resolvable signal in the X-nucleus spectrum, the depth of insertion into the lipid bilayer can be determined. The technique represents the adaptation of the Goldman-Shen 1 H spin-diffusion experiment with X-nucleus detection to proteins in hydrated lipid bilayers (>25% water by weight) in the gel state at 240 K. The experiments are demonstrated on the 21-kDa channel-forming domain of the toxin-like colicin E1 molecule incorporated into lipid vesicles. More than 32% of the protons in our sample are in mobile H 2 O molecules, which can be selected efficiently by the 1 H T 2 filter in the Goldman-Shen sequence. The transfer of 1 H magnetization from mobile H 2 O to the colicin E1 channel domain is 80% complete within only 5 ms. This transfer to the protein, probed by the amide 15 N signals, is faster than the transfer to the rigid protons on average, proving that most of the protein is preferentially located between the water and the lipid bilayer. From the spindiffusion and dipolar-dephasing data, 60% of the 24 lysine side groups are shown to be highly mobile. Quantitative depth profiling is demonstrated using the 31 P in the lipid phosphate head groups and the 13 C nuclei in the lipid acyl chains as distance markers for the spin diffusion.
Fluorine-19 NMR Chemical Shift Probes Molecular Binding to Lipid Membranes
The Journal of Physical Chemistry B, 2008
The binding of amphiphilic molecules to lipid bilayers is followed by 19 F NMR using chemical shift and line shape differences between the solution and membrane-tethered states of -CF 3 and -CHF 2 groups. A chemical shift separation of 1.6 ppm combined with a high natural abundance and high sensitivity of 19 F nuclei offers an advantage of using 19 F NMR spectroscopy as an efficient tool for rapid time-resolved screening of pharmaceuticals for membrane binding. We illustrate the approach with molecules containing both fluorinated tails and an acrylate moiety, resolving the signals of molecules in solution from those bound to synthetic dimyristoylphosphatidylcholine bilayers both with and without magic angle sample spinning. The potential in vitro and in vivo biomedical applications are outlined. The presented method is applicable with the conventional NMR equipment, magnetic fields of several Tesla, stationary samples, and natural abundance isotopes.
Novel NMR tools to study structure and dynamics of biomembranes
Chemistry and Physics of Lipids, 2002
Nuclear magnetic resonance (NMR) studies on biomembranes have benefited greatly from introduction of magic angle spinning (MAS) NMR techniques. Improvements in MAS probe technology, combined with the higher magnetic field strength of modern instruments, enables almost liquid-like resolution of lipid resonances. The cross-relaxation rates measured by nuclear Overhauser enhancement spectroscopy (NOESY) provide new insights into conformation and dynamics of lipids with atomic-scale resolution. The data reflect the tremendous motional disorder in the lipid matrix. Transfer of magnetization by spin diffusion along the proton network of lipids is of secondary relevance, even at a long NOESY mixing time of 300 ms. MAS experiments with re-coupling of anisotropic interactions, like the 13 C-1 H dipolar couplings, benefit from the excellent resolution of 13 C shifts that enables assignment of the couplings to specific carbon atoms. The traditional 2 H NMR experiments on deuterated lipids have higher sensitivity when conducted on oriented samples at higher magnetic field strength. A very large number of NMR parameters from lipid bilayers is now accessible, providing information about conformation and dynamics for every lipid segment. The NMR methods have the sensitivity and resolution to study lipid-protein interaction, lateral lipid organization, and the location of solvents and drugs in the lipid matrix.
International Journal of Molecular Sciences
The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A 19F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state 19F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profi...
Deuterium NMR and spin label ESR as probes of membrane organization
Journal of Colloid and Interface Science, 1977
A series of investigations of the mobility and organization of lipids in model and natural biological membranes using electron spin resonance (ESR) and nuclear magnetic resonance (NMR) of nitroxideand deuterium-labeled lipids, respectively, was performed. In most cases, parallel NMR and ESR experiments were carried out in order to compare the advantages and disadvantages of each technique. Both methods report the same qualitative picture of the lipid bilayer: high order and low mobility for acyl chain segmen ts near the carboxyl groups of the fatty acids and much less order and more rapid motion near the terminal methyl groups. However, the quantitative results differ markedly and it appears that ESR spin labels are unable to report the fine details of order and mobility gradients in lipid bilayers, possibly because of nitroxide-induced perturbations and the difficulty in resolving the spatial and temporal information from ESR spectra. The ESR method has the distinct advantages of superior sensitivity and speed where a qualitative answer is sufficient. In studies of deuteriumand nitroxide-labeled lipids in the plasma membrane of Acholeplasma laidlawii, both methods yield spectra containing characteristics of the gel-to-liquid crystal phase transition but only the NMR result is quantitatively consistent with the broad transition found from differential thermal analysis. The influence of cholesterol on acyl chain ordering in egg lecithin was studied as a function of the position of the deuterium or nitroxide label. Cholesterol increases the order parameter for all chain positions but the effect is greatest for positions 2-12. The 2H NMR results are discussed in terms of geometrical changes in the bilayer due to cholesterol. No evidence for a specific complex between lecithin and cholesterol is apparent. Multilamellar dispersions and single bilayer vesicles of phospholipids are compared as models for membrane processes: they yield equivalent information about mobility and order in the bilayers when the effects of vesicle overall rotation are correctly taken into account. many of them suffer from the difficult3' that
Interaction of Spin-Labeled Lipid Membranes with Transition Metal Ions
The Journal of Physical Chemistry B, 2015
The large values of spin relaxation enhancement (RE) for PC spinlabels in the phospholipid membrane induced by paramagnetic metal salts dissolved in the aqueous phase can be explained by Heisenberg spin exchange due to conformational fluctuations of the nitroxide group as a result of membrane fluidity, flexibility of lipid chains, and, possibly, amphiphilic nature of the nitroxide label. Whether the magnetic interaction occurs predominantly via Heisenberg spin exchange (Ni) or by the dipole−dipole (Gd) mechanism, it is essential for the paramagnetic ion to get into close proximity to the nitroxide moiety for efficient RE. For different salts of Ni the RE in phosphatidylcholine membranes follows the anionic Hofmeister series and reflects anion adsorption followed by anion-driven attraction of paramagnetic cations on the choline groups. This adsorption is higher for chaotropic ions, e.g., perchlorate. (A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules.) However, there is no anionic dependence of RE for model membranes made from negatively charged lipids devoid of choline groups. We used Ni-induced RE to study the thermodynamics and electrostatics of ion/membrane interactions. We also studied the effect of membrane composition and the phase state on the RE values. In membranes with cholesterol a significant difference is observed between PC labels with nitroxide tethers long enough vs not long enough to reach deep into the membrane hydrophobic core behind the area of fused cholesterol rings. This study indicates one must be cautious in interpreting data obtained by PC labels in fluid membranes in terms of probing membrane properties at different immersion depths when it can be affected by paramagnetic species at the membrane surface.