Phospholipids in Motion: High-Resolution 31P NMR Field Cycling Studies.

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 (R1). 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 R1 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.

Ca2+-independent binding of anionic phospholipids by phospholipase C δ1 EF-hand domain.

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.

Phosphatidylcholine "wobble" in vesicles assessed by high-resolution 13C field cycling NMR spectroscopy.

High resolution (13)C 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, R(1), observed down to 0.05 T is the result of dipolar and CSA relaxation components characterized by a single correlation time tau(c), with a small contribution from a faster motion contributing to CSA relaxation. At lower fields, R(1) increases further with a correlation time consistent with vesicle tumbling. The tau(c) is particularly interesting since it is 2-3 times slower than what is observed for (31)P of the same phospholipid. However, cholesterol increases the tau(c) for both (31)P and (13)C sites to the same value, approximately 25 ns. These observations suggest faster local motion dominates the dipolar relaxation of the (31)P, while a slower rotation or wobble dominates the relaxation of the carbonyl carbon by the alpha-CH(2) group. The faster motion must be damped with the sterol present. As a general methodology, high resolution (13)C field cycling may be useful for quantifying dynamics in other complex systems as long as a (13)C label (without attached protons) can be introduced.