Phospholipid bilayer surface configuration probed quantitatively by ³¹ P field-cycling NMR

Abstract

31 P relaxation of the diester phosphate of phospholipids in unilamellar vesicles has been studied from 0.004 to 11.7 T. Relaxation at very low fields, below 0.1 T, shows a rate increase that reflects a residual dipolar interaction with neighboring protons, probably dominated by the glycerol C3 protons. This interaction is not fully averaged by faster motion such as rotational diffusion perpendicular to the membrane surface. The remaining dipolar interaction, modulated by overall rotational diffusion of the vesicle and lateral diffusion of the lipid molecules, is responsible for the very low-field relaxation. These measurements yield a good estimate of the time-average angle between the membrane surface and the vector connecting the phosphorus to the glycerol C3 dynamics ͉ membranes ͉ phosphorus M uch remains to be known about the details of the configuration and dynamics of the phosphodiester region of phospholipid bilayer membranes, despite a large body of work (1, 2). Yet such knowledge is likely to be very useful in understanding how proteins and assemblies interact with this interfacial region. For example, there is indirect evidence that the peripheral membrane protein phosphatidylinositol-specific phospholipase inserts a tryptophan residue into a phosphatidylcholine membrane surface, which in turn activates the enzyme toward its substrates, but how this happens in detail is unknown (3, 4).The lack of structural information for phospholipid polar and interfacial moieties results from the difficulty in obtaining crystals and making other ordered structures that are necessary for most structural techniques. Even when available, the resemblance of such ordered structures to membranes functioning in vivo can be questioned. Modern methods of NMR spectroscopy are generally difficult to apply to this problem, mainly because of the slow rate of tumbling of molecules in reasonable analogs of biological membranes such as vesicles. Most NMR studies ( 1 H, 13 C, and 2 H) of membranes have focused on acyl chain dynamics (5-7). Phosphorus-31 NMR, which would appear ideally suited for obtaining information on the phosphodiester linkage conformation and dynamics, has seen limited use, because 31 P resonances in phospholipid aggregates exhibit a large linewidth due to their chemical-shift anisotropy (CSA) [although this property had made this nucleus very useful in characterizing the phase behavior of phospholipid bilayers (5)]. 31 P chemical shifts in phosphodiesters embedded in membranes do reflect orientation of chemical bonds relative to the membrane surface but not in a very usefully specific way. Structures deduced from other NMR data suffer from unknown dynamic averaging effects. Computer simulations show great promise to eventually give completely detailed information (8, 9) but still need to be validated with quantitative experiments.As a contribution to this problem, we have estimated the angle PH between the vector connecting the phosphorus to its nearest protons and the vector perpendicular to the membrane sur...

Insights into the Structural Specificity of the Cytotoxicity of 3-Deoxyphosphatidylinositols

“…25 In samples where phospholipid vesicles or large rod-shaped micelles form (e.g., diC 7 PC), there is a distinct rise in R 1 detected at very low fields (<0.06 T). 26 When well-separated from the nanosecond dispersion, a correlation time, τ v , that may reflect the contribution of particle tumbling to relaxation can be extracted. It has been proposed that τ v is equal to the rotational correlation time D r /6 where D r is the rotational diffusion constant of the individual lipid due to both diffusion of the entire aggregate, and translational diffusion of individual lipids relative to the aggregate.…”

“…It has been proposed that τ v is equal to the rotational correlation time D r /6 where D r is the rotational diffusion constant of the individual lipid due to both diffusion of the entire aggregate, and translational diffusion of individual lipids relative to the aggregate. 26 For micelles as opposed to vesicles, a detailed analysis is complicated by the fact that monomers and micelles coexist in fast exchange and that many micelles are usually rod-shaped rather than spherical. Nonetheless, the line shape of the R 1 versus field profile can indicate that micelles form, and whether they are large (such that a low field rise in R 1 is observed) or are small (in which case no low field rise is detected, but a minimum in rate at 1-2 T, suggesting a several ns correlation time).…”

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

“…To cycle the magnetic field, the sample is mechanically shuttled between the center high field region of the commercial NMR magnet's probe and a substantially lower magnetic field located above the probe before and after the delay times normally used in conventional NMR relaxation sequences (8). Analysis of the field dependence of spin lattice relaxation rates over a wide field range (0.002-11.7 T) provides several correlation times () for each phospholipid molecule on time scales ranging from ps to s and also allows us to separate dipolar and CSA interactions (8,9). Of particular interest is the intermediate [5][6][7][8][9][10] ns c that appears to be dominated by the wobbling of each phospholipid in the membrane (10).…”

“…Analysis of the field dependence of spin lattice relaxation rates over a wide field range (0.002-11.7 T) provides several correlation times () for each phospholipid molecule on time scales ranging from ps to s and also allows us to separate dipolar and CSA interactions (8,9). Of particular interest is the intermediate [5][6][7][8][9][10] ns c that appears to be dominated by the wobbling of each phospholipid in the membrane (10). This c provides a direct probe of lipid dynamics in these multicomponent membranes.…”

“…Thus, FCS allows us to screen different phospholipid compositions and to identify conditions that can be studied in detail by other techniques aimed at probing how protein binding alters the lipid environment. One such technique is high resolution field cycling 31 P NMR spectroscopy (fc-P-NMR) (8,9). Bacterial PI-PLCs recognize the headgroups of substrate, substrate analogue, and activator phospholipids, making 31 P NMR an obvious choice for studying protein-lipid interactions.…”