Solid-state NMR investigation of the selective disruption of lipid membranes by protegrin-1

The interaction of a beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), with various lipid membranes is investigated by (31)P, (2)H, and (13)C solid-state NMR. Mixed lipid bilayers containing anionic lipids and cholesterol are used to mimic the bacterial and mammalian cell membranes, respectively. (31)P and (2)H spectra of macroscopically oriented samples show that PG-1 induces the formation of an isotropic phase in anionic bilayers containing phosphatidylglycerol. Two-dimensional (31)P exchange experiments indicate that these isotropic lipids are significantly separate from the residual oriented lamellar bilayers, ruling out toroidal pores as the cause for the isotropic signal. (1)H spin diffusion experiments show that PG-1 is not exclusively bound to the isotropic phase but is also present in the residual oriented lamellar bilayers. This dynamic and morphological heterogeneity of the anionic membranes induced by PG-1 is supported by the fact that (13)C T(2) relaxation times measured under cross polarization and direct polarization conditions differ significantly. In contrast to the anionic membrane, the zwitterionic phosphatidylcholine (PC) membrane does not form an isotropic phase in the presence of PG-1 but shows significant orientational disorder. The addition of cholesterol to the PC bilayer significantly reduces this orientational disorder. The (13)C T(2) relaxation times of the PC lipids in the presence of both cholesterol and PG-1 suggest that the peptide may decrease the dynamic heterogeneity of the cholesterol-containing membrane. The observed selective interaction of PG-1 with different lipid membranes is consistent with its biological function and may be caused by its strong cationic and amphipathic structure.     

Solid-state NMR investigation of the selective perturbation of lipid bilayers by the cyclic antimicrobial peptide RTD-1

RTD-1 is a cyclic beta-hairpin antimicrobial peptide isolated from rhesus macaque leukocytes. Using (31)P, (2)H, (13)C, and (15)N solid-state NMR, we investigated the interaction of RTD-1 with lipid bilayers of different compositions. (31)P and (2)H NMR of uniaxially oriented membranes provided valuable information about how RTD-1 affects the static and dynamic disorder of the bilayer. Toward phosphatidylcholine (PC) bilayers, RTD-1 causes moderate orientational disorder independent of the bilayer thickness, suggesting that RTD-1 binds to the surface of PC bilayers without perturbing its hydrophobic core. Addition of cholesterol to the POPC membrane does not affect the orientational disorder. In contrast, binding of RTD-1 to anionic bilayers containing PC and phosphatidylglycerol lipids induces much greater orientational disorder without affecting the dynamic disorder of the membrane. These correlate with the selectivity of RTD-1 for anionic bacterial membranes as opposed to cholesterol-rich zwitterionic mammalian membranes. Line shape simulations indicate that RTD-1 induces the formation of micrometer-diameter lipid cylinders in anionic membranes. The curvature stress induced by RTD-1 may underlie the antimicrobial activity of RTD-1. (13)C and (15)N anisotropic chemical shifts of RTD-1 in oriented PC bilayers indicate that the peptide adopts a distribution of orientations relative to the magnetic field. This is most likely due to a small fraction of lipid cylinders that change the RTD-1 orientation with respect to the magnetic field. Membrane-bound RTD-1 exhibits narrow line widths in magic-angle spinning spectra, but the sideband intensities indicate rigid-limit anisotropies. These suggest that RTD-1 has a well-defined secondary structure and is likely aggregated in the membrane. These structural and dynamical features of RTD-1 differ significantly from those of PG-1, a related beta-hairpin antimicrobial peptide.     

Immobilization and aggregation of the antimicrobial peptide protegrin-1 in lipid bilayers investigated by solid-state NMR

The dynamics and aggregation of a beta-sheet antimicrobial peptide, protegrin-1 (PG-1), are investigated using solid-state NMR spectroscopy. Chemical shift anisotropies of F12 and V16 carbonyl carbons are uniaxially averaged in 1,2-dilauryl-sn-glycero-3-phosphatidylcholine (DLPC) bilayers but approach rigid-limit values in the thicker 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC) bilayers. The Calpha-Halpha dipolar coupling of L5 is scaled by a factor of 0.16 in DLPC bilayers but has a near-unity order parameter of 0.96 in POPC bilayers. The larger couplings of PG-1 in POPC bilayers indicate immobilization of the peptide, suggesting that PG-1 forms oligomeric aggregates at the biologically relevant bilayer thickness. Exchange NMR experiments on F12 (13)CO-labeled PG-1 show that the peptide undergoes slow reorientation with a correlation time of 0.7 +/- 0.2 s in POPC bilayers. This long correlation time suggests that in addition to aggregation, geometric constraints in the membrane may also contribute to PG-1 immobilization. The PG-1 aggregates contact both the surface and the hydrophobic center of the POPC bilayer, as determined by (1)H spin-diffusion measurements. Thus, solid-state NMR provides a wide range of information about the molecular details of membrane peptide immobilization and aggregation in lipid bilayers.     

Solid-state NMR measurements of the kinetics of the interaction between phospholamban and Ca2+-ATPase in lipid bilayers

Phospholamban (PLB) is a small transmembrane protein that regulates calcium transport across the sarcoplasmic reticulum (SR) of cardiac cells via a reversible inhibitory interaction with Ca2+-ATPase. In this work solid-state NMR methods have been used to investigate the dynamics of the inhibitory association between PLB and Ca2+-ATPase. Skeletal muscle Ca2+-ATPase was incorporated into phosphatidylcholine membranes together with a ten-fold excess of a null-cysteine mutant of PLB labelled with 13C at Leu-44 in the transmembrane domain ([alpha-13C-L44]AAA-PLB). In these membranes the PLB variant was found to partially inhibit Ca2+-ATPase by reducing the affinity of the enzyme for calcium. Cross-polarization magic angle spinning (CP-MAS) 13C NMR spectra of the membranes exhibited a signature peak from [alpha-13C-L44]AAA-PLB at 56 ppm. Changes in the intensity of the peak were observed at different temperatures, which was diagnostic of direct interaction between [alpha-13C-L44]AAA-PLB and Ca2+-ATPase. Measurements of dipolar couplings between the 13C label and neighbouring protons were analysed to show that the mean residency time for the association of AAA-PLB with Ca2+-ATPase was on the order of 2.5 ms at temperatures between 0 degrees C and 30 degrees C. This new NMR approach will be useful for examining how the association of the two proteins is affected by physiological stimuli such as kinases and the elevation of calcium concentration.     

Solid-state NMR chemical shift assignments of aquaporin Z in lipid bilayers

Aquaporin Z is the first identified prokaryotic water channel in Escherichia coli with a high water permeability and strict substrate selectivity. Here we report nearly complete (94% of amino acid residues) ¹³C and ¹⁵N chemical shift assignments of AqpZ reconstituted in the lipid bilayers using a set of 2D and 3D magic angle spinning solid-state NMR spectra. Secondary structure of AqpZ predicted from chemical shift assignments is generally similar to that of X-ray structure with a number of differences in loop and near-loop regions. The BMRB accession number of the assignments is 27244.     

Interaction of protegrin-1 with lipid bilayers: membrane thinning effect

Protegrins (PG) are important in defending host tissues, preventing infection via an attack on the membrane surface of invading microorganisms. Protegrins have powerful antibiotic abilities, but the molecular-level mechanisms underlying the interactions of their beta-sheet motifs with the membrane are not known. Protegrin-1 (PG-1) is composed of 18 amino acids with a high content of basic residues and two disulfide bonds. Here we focused on the stability of PG-1 at the amphipathic interface in lipid bilayers and on the details of the peptide-membrane interactions. We simulated all-atom models of the PG-1 monomer with explicit water and lipid bilayers composed of both homogeneous POPC (palmitoyl-oleyl-phosphatidylcholine) lipids and a mixture of POPC/POPG (palmitoyl-oleyl-phosphatidylglycerol) (4:1) lipids. We observed that local thinning of the lipid bilayers mediated by the peptide is enhanced in the lipid bilayer containing POPG, consistent with experimental results of selective membrane targeting. The beta-hairpin motif of PG-1 is conserved in both lipid settings, whereas it is highly bent in aqueous solution. The conformational dynamics of PG-1, especially the highly charged beta-hairpin turn region, are found to be mostly responsible for disturbing the membrane. Even though the eventual membrane disruption requires PG-1 oligomers, our simulations clearly show the first step of the monomeric effects. The thinning effects in the bilayer should relate to pore/channel formation in the lipid bilayer and thus be responsible for further defects in the membrane caused by oligomer.     

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.     

Conformational study of the protegrin-1 (PG-1) dimer interaction with lipid bilayers and its effect

Background  

Protegrin-1 (PG-1) is known as a potent antibiotic peptide; it prevents infection via an attack on the membrane surface of invading microorganisms. In the membrane, the peptide forms a pore/channel through oligomerization of multiple subunits. Recent experimental and computational studies have increasingly unraveled the molecular-level mechanisms underlying the interactions of the PG-1 β-sheet motifs with the membrane. The PG-1 dimer is important for the formation of oligomers, ordered aggregates, and for membrane damaging effects. Yet, experimentally, different dimeric behavior has been observed depending on the environment: antiparallel in the micelle environment, and parallel in the POPC bilayer. The experimental structure of the PG-1 dimer is currently unavailable.     

Solid-state NMR analysis comparing the designer-made antibiotic MSI-103 with its parent peptide PGLa in lipid bilayers

The amphiphilic alpha-helical peptide (KIAGKIA)3-NH2 (MSI-103) is a designer-made antibiotic, based on the natural sequence of PGLa from Xenopus laevis. Here, we have characterized the concentration-dependent alignment and dynamic behavior of MSI-103 in lipid membranes by solid-state 2H and 19F NMR, using orientational constraints from seven Ala-d3-labeled analogues and five 4-CF3-phenylglycine labels. As previously found for PGLa, MSI-103, too, assumes a flat surface-bound S-state alignment at low peptide concentrations, and it also realigns to a tilted T-state at higher concentrations. For PGLa, the stability of the T-state had been attributed to the specific assembly of antiparallel dimers; hence, it is remarkable that the artificial KIAGKIA repeat sequence can also dimerize in the same way in liquid crystalline lipid bilayers. Oriented circular dichroism analysis shows that for MSI-103 the threshold for realignment from the S-state to the T-state is approximately 3-fold lower than for PGLa (at a peptide-to-lipid ratio of 1:240 in dimyristoylphosphatidylcholine, compared to 1:80). Furthermore, MSI-103 becomes laterally immobilized in the lipid bilayer at a concentration ratio of 1:50, which occurs for PGLa only above 1:20. The superior antimicrobial activity of MSI-103 over PGLa thus appears to correlate with its stronger tendency to realign and self-assemble. The hemolytic activities of MSI-103 and its analogues, on the other hand, are shown here to correlate purely with the respective changes in hydrophobicity.     

Deuterium NMR investigation of an amphotericin B derivative in mechanically aligned lipid bilayers

The methyl-d(3) amide derivative of the polyene antibiotic amphotericin B was synthesized, assayed for biological activity, incorporated into mechanically aligned bilayers of dipalmitoylphosphatidylcholine (DPPC), and examined by deuterium and phosphorus NMR. The amide derivative has a lesser, but qualitatively similar, biological activity relative to amphotericin B. Incorporation of the amide derivative and ergosterol into aligned DPPC bilayers resulted in a single, stable bilayer phase, as shown by phosphorus NMR of the DPPC headgroups. Deuterium NMR spectra revealed one major (2)H quadrupolar splitting and one major (2)H-(1)H dipolar splitting in the liquid-crystalline phase, consistent with a high degree of alignment and a single, averaged physical state for amphotericin B methyl-d(3) amide in the bilayer. Variations of the quadrupolar and dipolar splittings as a function of macroscopic sample orientation and temperature indicated that the amide derivative undergoes fast rotation about a motional axis that is parallel to the bilayer normal.     

Phospholamban forms Ca2+-selective channels in lipid bilayers

Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. A role for phospholamban in the control of Ca2+ transport by the sarcoplasmic reticulum has been postulated, but the mechanism is incompletely understood. Structural characterization of the purified protein suggests that it is capable of forming a membrane-spanning pore (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). The experiments described here tested the hypothesis that canine cardiac phospholamban, isolated in the fully dephosphorylated state, forms ion channels in lipid bilayers. Phospholamban purified by two different methods formed channels that were permeable to cations, exhibited spontaneous openings and closings, and were selective for Ca2+ over K+. Dihydropyridine drugs and ryanodine did not affect channel activity. The putative membrane-spanning portion of the molecule, residues 26-52, also formed channels in the bilayer. The putative regulatory portion of the molecule, residues 2-25, did not. The results suggest that phospholamban may regulate sarcoplasmic reticulum Ca2+ flux by acting as a Ca2+ channel.     

SNARE complex alters the interactions of the Ca2+ sensor synaptotagmin 1 with lipid bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca²⁺ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca²⁺ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca²⁺-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca²⁺ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.     

Solid-state NMR and MD simulations of the antiviral drug amantadine solubilized in DMPC bilayers

The interactions of ¹⁵N-labeled amantadine, an antiinfluenza A drug, with DMPC bilayers were investigated by solid-state NMR and by a 12.6-ns molecular dynamics (MD) simulation. The drug was found to assume a single preferred orientation and location when incorporated in these bilayers. The experimental and MD computational results demonstrate that the long axis of amantadine is on average parallel to the bilayer normal, and the amine group is oriented toward the headgroups of the lipid bilayers. The localization of amantadine was determined by paramagnetic relaxation and by the MD simulation showing that amantadine is within the interfacial region and that the amine interacts with the lipid headgroup and glycerol backbone, while the hydrocarbon portion of amantadine interacts with the glycerol backbone and much of the fatty acyl chain as it wraps underneath the drug. The lipid headgroup orientation changes on drug binding as characterized by the anisotropy of ³¹P chemical shielding and ¹⁴N quadrupolar interactions and by the MD simulation.     

Structural dynamics and topology of phospholamban in oriented lipid bilayers using multidimensional solid-state NMR

Phospholamban (PLN), a single-pass membrane protein, regulates heart muscle contraction and relaxation by reversible inhibition of the sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Studies in detergent micelles and oriented lipid bilayers have shown that in its monomeric form PLN adopts a dynamic L shape (bent or T state) that is in conformational equilibrium with a more dynamic R state. In this paper, we use solid-state NMR on both uniformly and selectively labeled PLN to refine our initial studies, describing the topology and dynamics of PLN in oriented lipid bilayers. Two-dimensional PISEMA (polarization inversion spin exchange at the magic angle) experiments carried out in DOPC/DOPE mixed lipid bilayers reveal a tilt angle of the transmembrane domain with respect to the static magnetic field, of 21 +/- 2 degrees and, at the same time, map the rotation angle of the transmembrane domain with respect to the bilayer. PISEMA spectra obtained with selectively labeled samples show that the cytoplasmic domain of PLN is helical and makes an angle of 93 +/- 6 degrees with respect to the bilayer normal. In addition, using samples tilted by 90 degrees , we find that the transmembrane domain of PLN undergoes fast long-axial rotational diffusion about the bilayer normal with the cytoplasmic domain undergoing this motion and other complex dynamics, scaling the values of chemical shift anisotropy. While this dynamic was anticipated by previous solution NMR relaxation studies in micelles, these measurements in the anisotropic lipid environment reveal new dynamic and conformational features encoded in the free protein that might be crucial for SERCA recognition and subsequent inhibition.     

Solid-state NMR structure determination of melittin in a lipid environment

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.     

Inward rectification of the IRK1 K+ channel reconstituted in lipid bilayers.

Inwardly rectifying potassium (K+) channels (IRK1) were incorporated into lipid bilayers to test the relative contributions of various mechanisms to inward rectification. IRK1 channels were expressed in Xenopus laevis oocytes and oocyte membrane vesicles containing the channels were fused with lipid bilayers. The major properties of the IRK1 channel were similar whether measured in the oocyte membrane or lipid bilayer; the single channel conductance was 21 pS in 140 mM symmetrical [K+] and varied as a square root of external [K+]. Importantly, IRK1 channels display voltage-dependent inward rectification in the absence of divalent ions or charged regulators such as spermine, indicating that they possess an intrinsic rectification mechanism. Although rectification was significantly increased by either Mg2+ or spermine added to the cytoplasmic face of the channel, their effects could not be explained by simple block of the open pore. The Hille and Schwartz (1978) model, originally proposed to explain inward rectification by singly charged blocking particles, cannot be used to explain rectification by multiply charged blocking particles. As an alternative, we propose that in addition to a slow gating mechanism producing long lasting open and closed states, there is a distinct, intrinsic fast gating process amplified by cytoplasmic Mg2+ and/or polyamine binding to the channel.     

Solid-state NMR investigation of indomethacin-cyclodextrin complexes in PEG 6000 carrier

Various solid dispersions of alpha-, beta- and gamma-cyclodextrin (CD) in PEG 6000 with and without the addition of 5% w/w indomethacin were prepared by the melting method using the original components. The samples were investigated by solid-state (13)C NMR, and the interactions between the drug and the cyclodextrins were evaluated. The indomethacin-gamma-CD phase with tetragonal symmetry found in a previous X-ray study gave chemical shifts which suggested that this phase is a complex between indomethacin and gamma-CD. Evidence of an indomethacin-beta-CD complex were found. A distribution of the chemical shifts for beta-CD was attributed to the possible formation of different types of complexes between indomethacin and beta-CD. No complex formation was found in the alpha-CD system. The degree of relative crystallinity of the samples in the gamma-CD system was measured by (1)H NMR, X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), and modulated-temperature DSC (MTDSC). The results obtained by the NMR, XRD, and DSC techniques showed that the dispersions were less crystalline than the pure polymer carrier, and the dispersion containing the indomethacin-gamma-CD complex had the lowest degree of crystallinity. By the MTDSC method a deviation was found for the PEG 6000/indomethacin dispersion. This emphasizes that the different techniques give specific information on the crystallinity.     

Actin modifies Ca2+ block of epithelial Na+ channels in planar lipid bilayers

The mechanism by which the cytoskeletal protein actin affects the conductance of amiloride-sensitive epithelial sodium channels (ENaC) was studied in planar lipid bilayers. In the presence of monomeric actin, we found a decrease in the single-channel conductance of alpha-ENaC that did not occur when the internal [Ca2+]free was buffered to <10 nM. An analysis of single-channel kinetics demonstrated that Ca2+ induced the appearance of long-lived closed intervals separating bursts of channel activity, both in the presence and in the absence of actin. In the absence of actin, the duration of these bursts and the time spent by the channel in its open, but not in its short-lived closed state, were inversely proportional to [Ca2+]. This, together with a lengthening of the interburst intervals, translated into a dose-dependent decrease in the single-channel open probability. In contrast, a [Ca2+]-dependent decrease in alpha-ENaC conductance in the presence of actin was accompanied by lengthening of the burst intervals with no significant changes in the open or closed (both short- and long-lived) times. We conclude that Ca2+ acts as a "fast-to-intermediate" blocker when monomeric actin is present, producing a subsequent attenuation of the apparent unitary conductance of the channel.     

Solid-state H and N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca²⁺ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca²⁺ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with ²H and ¹⁵N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). ²H and ¹⁵N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD₃-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca²⁺-ATPase; thus, showing increased inhibition of Ca²⁺-ATPase.     

Orientation dependence of NMR relaxation time, T(1rho), in lipid bilayers

The orientation dependence of the low frequency NMR relaxation time, T(1rho), of protons in aligned phospholipid bilayers was measured using 13C cross polarisation and direct proton experiments. The contribution of intra- and inter-molecular interactions to proton T(1rho) was determined by using dimyristoyl phosphatidylcholine (DMPC) with one hydrocarbon chain deuterated and dispersed in perdeuterated DMPC. The results indicated that intramolecular motions on the kHz timescale were the major cause of T(1rho) relaxation in phospholipid bilayers.