High resolution 13C NMR field cycling (covering 11.7 down to 0.002 T) relaxation studies of the sn-2 carbonyl of phosphatidylcholines in vesicles provide a detailed look at the dynamics of this position of the phospholipid in vesicles. The spin-lattice relaxation rate, R1, observed down to 0.05 T is the result of dipolar and CSA relaxation components characterized by a single correlation time τc, with a small contribution from a faster motion contributing CSA relaxation. At lower fields, R1 increases further with a correlation time consistent with vesicle tumbling. The τc is particularly interesting since it is 2-3 times slower than what is observed for 31P of the same phospholipid. However, cholesterol increases the τc for both 31P and 13C sites to the same value, ~25 ns. These observations suggest faster local motion dominates the dipolar relaxation of the 31P while a slower rotation or wobble dominates the relaxation of the carbonyl carbon by the α-CH2 group. The faster motion must be damped with the sterol present. As a general methodology, high resolution 13C field cycling may be useful for quantifying dynamics in other complex systems as long as a 13C label (without attached protons) can be introduced.
Background:The conserved EF-hand (EF) domain is necessary for active phospholipase. Results: EF binds to anionic phospholipid-containing vesicles; EF mutations introduced into PLC ␦1 reduce activity not recoverable with added PIP 2 . Conclusion: EF-hand domain aids substrate binding in the active site when the protein is membrane-anchored. Significance: This may be the function of the EF-hand domain in other PLC enzymes as well.
Diverse phospholipid motions are key to membrane function but can be quite difficult to untangle and quantify. High-resolution field cycling 31P NMR spin-lattice relaxometry, where the sample is excited at high field, shuttled in the magnet bore for low-field relaxation, then shuttled back to high field for readout of the residual magnetization, provides data on phospholipid dynamics and structure. This information is encoded in the field dependence of the 31P spin-lattice relaxation rate (R 1). In the field range from 11.74 down to 0.003 T, three dipolar nuclear magnetic relaxation dispersions (NMRDs) and one due to 31P chemical shift anisotropy contribute to R 1 of phospholipids. Extraction of correlation times and maximum relaxation amplitudes for these NMRDs provides (1) lateral diffusion constants for different phospholipids in the same bilayer, (2) estimates of how additives alter the motion of the phospholipid about its long axis, and (3) an average 31P–1H angle with respect to the bilayer normal, which reveals that polar headgroup motion is not restricted on a microsecond timescale. Relative motions within a phospholipid are also provided by comparing 31P NMRD profiles for specifically deuterated molecules as well as 13C and 1H field dependence profiles to that of 31P. Although this work has dealt exclusively with phospholipids in small unilamellar vesicles, these same NMRDs can be measured for phospholipids in micelles and nanodisks, making this technique useful for monitoring lipid behavior in a variety of structures and assessing how additives alter specific lipid motions.
High resolution field cycling <sup>31</sup>P NMR spin-lattice relaxometry, where the sample is excited at high field, shuttled in the magnet bore for low field relaxation, then shuttled back to high field for readout of the residual magnetization, provides data on phospholipid dynamics and structure. In the field range from 11.74 down to 0.003 T three dipolar nuclear magnetic relaxation dispersions (NMRDs) and one due to <sup>31</sup>P chemical shift anisotropy contribute to R<sub>1 </sub>of phospholipids<sub>.</sub> Extraction of correlation times and maximum relaxation amplitudes for these NMRDs provides (1) lateral diffusion constants for different phospholipids in the same bilayer (illustrated with phospholipase C binding), (2) estimates of how additives alter the motion of the phospholipid about its long axis (looking at cholesterol effects), and (3) an average <sup>31</sup>P – <sup>1</sup>H angle with respect to the bilayer normal, which reveals that polar head group motion is not restricted on a µs timescale. Although this deals exclusively with phospholipids in small unilamellar vesicles, these same NMRDs can be measured for phospholipids in micelles and nanodiscs, making this technique useful for monitoring lipid behavior in a variety of structures.
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