Paul J. R. Spooner

PHOSPHOLIPID PHASE TRANSITIONS AS REVEALED BY NMR

Aqueous dispersions of phospholipids can adopt a range of polymorphic phases which include bilayer and non-bilayer forms. Within the bilayer form, laterally separated phases may be induced as a result of surface electrostatic associations, thermotropic behaviour, lipid-protein interactions or because of molecular mismatch between chemically distinct phospholipids. Nuclear magnetic resonance (NMR) methods, designed to exploit the properties of either indigenous nuclei or isotopic labels introduced specifically into a phospholipid, can be used in some cases to describe the molecular properties and behaviour of phospholipids in both macroscopically distinct phases and in molecularly distinct phases within the same polymorphic state. If the molecular motion of phospholipids in co-existing phases is sufficiently different, NMR methods can, in principle, give estimates of the life-time of the phases and the rate of molecular exchange between the phases.

Rotational Mobility and Orientational Stability of a Transport Protein in Lipid Membranes

A single-cysteine mutant of the lactose transport protein LacS(C320A/W399C) from Streptococcus thermophilus was selectively labeled with a nitroxide spin label, and its mobility in lipid membranes was studied as a function of its concentration in the membrane by saturation-transfer electron spin resonance. Bovine rhodopsin was also selectively spin-labeled and studied to aid the interpretation of the measurements. Observations of spin-labeled proteins in macroscopically aligned bilayers indicated that the spin label tends to orient so as to reflect the transmembrane orientation of the protein. Rotational correlation times of 1-2 micros for purified spin-labeled bovine rhodopsin in lipid membranes led to viscosities of 2.2 poise for bilayers of dimyristoylphosphatidylcholine (28 degrees C) and 3.0 poise for the specific mixture of lipids used to reconstitute LacS (30 degrees C). The rotational correlation time for LacS did not vary significantly over the range of low concentrations in lipid bilayers, where optimal activity was seen to decrease sharply and was determined to be 9 +/- 1 micros (mean +/- SD) for these samples. This mobility was interpreted as being too low for a monomer but could correspond to a dimer if the protein self-associates into an elongated configuration within the membrane. Rather than changing its oligomeric state, LacS appeared to become less ordered at the concentrations in aligned membranes exceeding 1:100 (w/w) with respect to the lipid.

A distance measurement between specific sites on the cytoplasmic surface of bovine rhodopsin in rod outer segment disk membranes.

Structural information on mammalian integral membrane proteins is scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation with rhodopsin kinase. Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label. Magic angle spinning 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.

Membrane protein structure determination by solid state NMR

In the last 3–5 years, solid state NMR has matured as a method for defining structural details of membrane-embedded proteins and peptides. It is still not, and may never be, a method of choice for complete structural resolution, but the power of the approach for certain systems, and in resolving details for binding sites of ligands and specific parts of proteins, is being proved. As with solution state NMR, stable NMR isotope (2H, 13C, 15N, 19F) incorporation into such large and complex biomolecules has made a tremendous impact in the area, aiding spectral assignments, increasing the amount and value of the information gained, and permitting identification of specific functional intermediates. The practical details of handling membrane proteins, which has for so long dogged every structural and biochemical study of membrane proteins, including crystallography, are not absent in the solid state NMR approach. It is simply that the protein can be studied in situ, or at least in reconstituted systems when the purified or isolated proteins can be reintroduced into a membrane for study. In addition, the molecular weight limit (Mr < 30 000) of solution state NMR (due to the slow molecular tumbling rate with respect to the applied field), is not limiting with solid state methods. Solid state NMR methods can now be used to study membrane proteins and peptides, but support from chemical, molecular biological and biochemical approaches are required to produce systems suitable for giving the desired molecular information. Specialized instrumentation is also required (high power NMR probes with spinning or static capability, specific pulse methods, good temperature stability and wide bore magnets) and some applications can present a challenge experimentally in setting up the NMR instrument. However, the method is emerging as a useful addition for the structural biologist.

Weak substrate binding to transport proteins studied by NMR.

The weak binding of sugar substrates fails to induce any quantifiable physical changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays. Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection. Cross-polarization from proton to carbon spins could detect the portion of 13C-labeled substrate associated with 0.2 micromol of the functional transport system overexpressed in the native membranes. The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates L-fucose and L-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer D-fucose. FucP appeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10(-1) s), although its dynamics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system.

Dynamics in a protein-lipid complex: nuclear magnetic resonance measurements on the headgroup of cardiolipin when bound to cytochrome c.

Deuterium and phosphorus nuclear magnetic resonance (NMR) has been used to investigate the dynamics of slow motional processes induced in bilayer cardiolipin upon binding with cytochrome c. 31P NMR line shapes suggest that protein binding induces less restricted, isotropic-like motions in the lipid phosphates within the ms time scale of this measurement. However, these motions impart rapid transverse relaxation to methylene deuterons adjacent to the phosphate in the lipid headgroup and so did not feature strongly in the NMR line shapes recorded from these nuclei by using the quadrupolar echo. Nonetheless, motional characteristics of the headgroup deuterons were accessible to a dynamic NMR approach using the Carr-Purcell-Meiboom-Gill multiple-pulse experiment. Compared to the well-studied case of deuterons in fatty acyl chains of bilayer phosphatidylcholine, the motions determining the 2H spin transverse relaxation in the headgroup of bilayer cardiolipin were much faster, having a lower limit in the 5-10 kHz range. On binding with cytochrome c, the T2 effecting motions in the cardiolipin headgroup became faster still, with rates comparable to the residual quadrupolar coupling frequency of the headgroup deuterons (approximately 25 kHz) and so coincided with the time scale for recording the quadrupolar echo (approximately 40 microseconds). It is concluded that the headgroup of cardiolipin does not exclusively report localized dynamic information but is particularly sensitive to collective motions occurring throughout the bilayer molecules. Although the rates of collective modes of motion may be dependent on the lipid type in pure lipid bilayers, these low-frequency fluctuations appear to occupy a similar dynamic range in a variety of lipid-protein systems, including the natural membranes.

Synthesis of optically active polyunsaturated diacylglycerols

Abstract 1,2-Isopropylidene-3-methoxyethoxymethyl- sn -glycerol ( 2 ) is used to access complex polyunsaturated diacylglycerols of high optical purity such as 1,2-dilinoleoyl- sn -glycerol ( 5 ). Using a similar methodology its enantiomer, 2,3-dilinoleoyl- sn -glycerol, can be obtained from 1-methoxyethoxymethyl-2,3-isopropylidene- sn -glycerol.

31P-CP–MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE

The binding of tetraphenylphosphonium (TPP+) to EmrE, a membrane‐bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross‐polarisation–magic‐angle spinning nuclear magnetic resonance spectroscopy (CP–MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP–MAS could selectively distinguish binding of TPP+ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.

Synthesis of isotopically labelled cardiolipins

Abstract A phosphotriester approach is used to access the complex phospholipid, cardiolipin (1) with polyunsaturated fatty acid chains. The synthetic method allows the specific incorporation of isotopic labels within the molecule and sets up the desired configuration at all three chiral centres which corresponds to the configuration of the natural phospholipid. Using such a methodology a range of pure optically active cardiolipin molecular species have been synthesized.

Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Abstract Using solid-state NMR approaches, it is now possible to define the structure and dynamics of binding for a small, isotopically (2H, 13C, 19F, 15N) labelled ligand, prosthetic group or solute in its binding site of a membrane-bound protein at near physiological conditions in natural membrane fragments or in reconstituted complexes. Studies of oriented membranes permit the orientational bond vectors of labelled groups to be determined to good precision, as shown for retinal in bacteriorhodopsin and bovine rhodopsin. Using novel magic angle spinning NMR methods on membrane dispersions, high-resolution NMR spectra can be obtained. Dipolar couplings can be reintroduced into the spectrum of labelled ligands in their binding sites of membrane-bound proteins to give interatomic distances to high precision (±0.5 Å). Relaxation and cross-polarization data give estimates for the kinetics for on-off rates for binding. In addition, chemical shifts can be measured directly to help provide details of the binding environment for a bound ligand, as shown for analogues of drugs used in peptic ulcer treatment in the gastric ATPase, and for acetylcholine in the acetylcholine receptor.