Alfred G. Redfield

Stimulated echo NMR spectra and their use for heteronuclear two-dimensional shift correlation

Abstract NMR spectra are obtained from Fourier transformation of the latter part of a Hahn stimulated echo. The sequence is 90°-τ a -90°-τ b -90°-τ a -digitize versus τ 2 . This is demonstrated for transfer RNA in H 2 O using the semiselective J ® pulse (90°-τ p -90°), developed by Plateau and Gueron in place of each 90° pulse. 2D correlation between 13 N and protons is obtained by applying a 90°-τ 1 -90° 15 N sequence during τ b and 15 N decoupling while digitizing. The second Fourier transform is performed with respect to τ 1 after discarding the imaginary parts of the first transform, to obtain a real 2D map. Such a 2D spectrum has been obtained for 5 m M 15 NH 4 Cl and several other small molecules. The sequence should be useful for studies of 15 N labeled macromolecules.

Rotation of Lipids in Membranes: Molecular Dynamics Simulation, 31P Spin-Lattice Relaxation, and Rigid-Body Dynamics

Molecular dynamics simulations and (31)P-NMR spin-lattice (R(1)) relaxation rates from 0.022 to 21.1 T of fluid phase dipalmitoylphosphatidylcholine bilayers are compared. Agreement between experiment and direct prediction from simulation indicates that the dominant slow relaxation (correlation) times of the dipolar and chemical shift anisotropy spin-lattice relaxation are approximately 10 ns and 3 ns, respectively. Overall reorientation of the lipid body, consisting of the phosphorus, glycerol, and acyl chains, is well described within a rigid-body model. Wobble, with D(perpendicular)= 1-2 x 10(8) s(-1), is the primary component of the 10 ns relaxation; this timescale is consistent with the tumbling of a lipid-sized cylinder in a medium with the viscosity of liquid hexadecane. The value for D(parallel), the diffusion constant for rotation about the long axis of the lipid body, is difficult to determine precisely because of averaging by fast motions and wobble; it is tentatively estimated to be 1 x 10(7) s(-1). The resulting D(parallel)/D( perpendicular) approximately 0.1 implies that axial rotation is strongly modulated by interactions at the lipid/water interface. Rigid-body modeling and potential of mean force evaluations show that the choline group is relatively uncoupled from the rest of the lipid. This is consistent with the ratio of chemical shift anisotropy and dipolar correlation times reported here and the previous observations that (31)P-NMR lineshapes are axially symmetric even in the gel phase of dipalmitoylphosphatidylcholine.

Proton NMR and NOE structural and dynamic studies of larger proteins and nucleic acids aided by isotope labels: T4 lysozyme.

This article reviews methods based on direct observation of proton NMR in macromolecules containing 13C or 15N labels. The resonances and Overhauser effects of protons attached to the labels can be edited or filtered from the remaining overlapping resonances. This leads to simplification of the spectra when labels are incorporated selectively. In 2D and related methods the labels chemical shift provides a second dimension which is useful for spectral differentiation and identification. The methods are useful for larger proteins and we describe our progress on studies of T4 lysozyme, mass 18.7 kD, in which we have already identified a large number of resonances.

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

D-3-deoxyphosphatidylinositol (D-3-deoxy-PI) derivatives have cytotoxic activity against various human cancer cell lines. These phosphatidylinositols have a potentially wide array of targets in the phosphatidylinositol-3-kinase (PI3K)/Akt signaling network. To explore the specificity of these types of molecules, we have synthesized D-3-deoxydioctanoylphosphatidylinositol (D-3-deoxy-diC8PI), D-3,5-dideoxy-diC8PI, and D-3-deoxy-diC8PI-5-phosphate and their enantiomers, characterized their aggregate formation by novel high-resolution field cycling (31)P NMR, and examined their susceptibility to phospholipase C (PLC), their effects on the catalytic activities of PI3K and PTEN against diC8PI and diC8PI-3-phosphate substrates, respectively, and their ability to induce the death of U937 human leukemic monocyte lymphoma cells. Of these molecules, only D-3-deoxy-diC8PI was able to promote cell death; it did so with a median inhibitory concentration of 40 microM, which is much less than the critical micelle concentration of 0.4 mM. Under these conditions, little inhibition of PI3K or PTEN was observed in assays of recombinant enzymes, although the complete series of deoxy-PI compounds did provide insights into ligand binding by PTEN. D-3-deoxy-diC8PI was a poor substrate and not an inhibitor of the PLC enzymes. The in vivo results are consistent with the current thought that the PI analogue acts on Akt1, since the transcription initiation factor eIF4e, which is a downstream signaling target of the PI3K/Akt pathway, exhibited reduced phosphorylation on Ser209. Phosphorylation of Akt1 on Ser473 but not Thr308 was reduced. Since the potent cytotoxicity for U937 cells was completely lost when L-3-deoxy-diC8PI was used as well as when the hydroxyl group at the inositol C5 in D-3-deoxy-diC8PI was modified (by either replacing this group with a hydrogen or phosphorylating it), both the chirality of the phosphatidylinositol moiety and the hydroxyl group at C5 are major determinants of the binding of 3-deoxy-PI to its target in cells.

Defining Specific Lipid Binding Sites for a Peripheral Membrane Protein in Situ Using Subtesla Field-cycling NMR

Despite the profound physiological consequences associated with peripheral membrane protein localization, only a rudimentary understanding of the interactions of proteins with membrane surfaces exists because these questions are inaccessible by commonly used structural techniques. Here, we combine high resolution field-cycling 31P NMR relaxation methods with spin-labeled proteins to delineate specific interactions of a bacterial phospholipase C with phospholipid vesicles. Unexpectedly, discrete binding sites for both a substrate analogue and a different phospholipid (phosphatidylcholine) known to activate the enzyme are observed. The lifetimes for the occupation of these sites (when the protein is anchored transiently to the membrane) are >1–2 μs (but <1 ms), which represents the first estimate of an off-rate for a lipid dissociating from a specific site on the protein and returning to the bilayer. Furthermore, analyses of the spin-label induced NMR relaxation corroborates the presence of a discrete tyrosine-rich phosphatidylcholine binding site whose location is consistent with that suggested by modeling studies. The methodology illustrated here may be extended to a wide range of peripheral membrane proteins.

Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity INSIGHTS INTO PHOSPHATIDYLCHOLINE ACTIVATION AND SURFACE DILUTION INHIBITION

The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (τc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased τc. PI-PLC activity and phospholipid τc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC τc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.

Modified Hartmann–Hahn double NMR in solids for high resolution at low gyromagnetic ratio: CaF2 and quadrupole interaction in MgF2

Moderately high resolution NMR of rare spins in solids is described, using a modified Hartmann–Hahn double resonance experiment. Resolution is achieved by high power resonant decoupling of abundant spins, and the Hartmann–Hahn condition is achieved by irradiation of the rare spins off resonance, which preserves their chemical shift in the rotating frame. The rare spins are saturated with an additional weak variable frequency rf field. Following this cross‐polarization period is a period when strong gated rf is applied off resonance to the abundant spins. This permits time‐shared observation of the abundant spin magnetization, which reflects saturation of the rare system during the previous cycle. At the same time it serves to replenish the rare spin magnetization because it is off resonance. Transition between the two modes is adiabatic. This method distorts the rare‐spin line shape, but a related model experiment is also described which would not do so. This method is expected to be more useful than that...

Observation of the phosphatidyl ethanolamine amino proton magnetic resonance in phospholipid vesicles: Inside/outside ratios and proton transport☆

The amino proton resonance of phosphatidyl ethanolamine in sonicated mixed phospholipid vesicles is observed 3.3 ppm downfield from H2O. Above pH ∼ 5 it is broadened beyond detectability as a result of exchange with H2O protons. In low salt, resonances of amino protons inside the vesicles appear to persist as the pH is raised, while those on the outside disappear. Solvent catalized proton conduction along the surface is proposed, with an effective -NH2 to -NH3 transfer rate of about 8 × 105 sec−1 at 25°C.

Phospholipid-binding Sites of Phosphatase and Tensin Homolog (PTEN) EXPLORING THE MECHANISM OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE ACTIVATION

Background: PTEN activity is enhanced by PI(4,5)P2 via either an allosteric or interfacial effect. Results: 31P field cycling NMR with spin-labeled PTEN detects a PI(4,5)P2-binding site near but not in the active site. Conclusion: A model for PTEN identifies Lys-13 and Arg-47 as part of the PI(4,5)P2 site. Significance: Occupation of the PI(4,5)P2-binding site on PTEN stabilizes productive interfacial binding. The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide 31P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling 31P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P2 binds on the protein. The results are used to model a discrete site for a PI(4,5)P2 molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P2 site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P2 near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis.

Phospholipid Reorientation at the Lipid/Water Interface Measured by High Resolution 31P Field Cycling NMR Spectroscopy

The magnetic field dependence of the 31P spin-lattice relaxation rate, R1, of phospholipids can be used to differentiate motions for these molecules in a variety of unilamellar vesicles. In particular, internal motion with a 5- to 10-ns correlation time has been attributed to diffusion-in-a-cone of the phosphodiester region, analogous to motion of a cylinder in a liquid hydrocarbon. We use the temperature dependence of 31P R1 at low field (0.03-0.08 T), which reflects this correlation time, to explore the energy barriers associated with this motion. Most phospholipids exhibit a similar energy barrier of 13.2 +/- 1.9 kJ/mol at temperatures above that associated with their gel-to-liquid-crystalline transition (Tm); at temperatures below Tm, this barrier increases dramatically to 68.5 +/- 7.3 kJ/mol. This temperature dependence is broadly interpreted as arising from diffusive motion of the lipid axis in a spatially rough potential energy landscape. The inclusion of cholesterol in these vesicles has only moderate effects for phospholipids at temperatures above their Tm, but significantly reduces the energy barrier (to 17 +/- 4 kJ/mol) at temperatures below the Tm of the pure lipid. Very-low-field R1 data indicate that cholesterol inclusion alters the averaged disposition of the phosphorus-to-glycerol-proton vector (both its average length and its average angle with respect to the membrane normal) that determines the 31P relaxation.