Anthony Watts

Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from the rectal gland of Squalus acanthias have been studied by using spin-labeled lipids in conjunction with electron spin resonance (ESR) spectroscopy. Lipid-protein associations are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component which corresponds very closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled lipids whose motion is very significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A stoichiometry of approximately 66 lipids per 265 000-dalton protein is found for the motionally restricted component of those spin-labeled lipids (e.g., phosphatidylcholine) which show least specificity for the protein. This corresponds approximately to the number of lipids which may be accommodated within the first shell around the alpha 2 beta 2 protein dimer. A selectivity of the various spin-labeled lipids for the motionally restricted component associated with the protein is found in the following order: cardiolipin greater than phosphatidylserine approximately stearic acid greater than or equal to phosphatidic acid greater than phosphatidylglycerol approximately phosphatidylcholine approximately phosphatidylethanolamine approximately androstanol.

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.

A simple but efficient 13C MAS NMR method is presented for the determination of the location of embedded molecules such as peptides relative to biological membrane surfaces by exploiting the interaction with paramagnetic lanthanide ions. Using various aqueous Dy3+ concentrations a distance-dependent differential paramagnetic quenching of NMR lipid resonance intensities for specific carbon sites was observed, with residues at the bilayer surface quenched effectively and hydrophobic sites unaffected by Dy3+. Tested on the membrane-embedded 50 residue long M13 coat protein, 13C labeled at its Val-29 and Val-31 residues, no paramagnetic quenching was observed for the peptide resonances by Dy3+, suggesting that Val-29 and Val-31 are not in close proximity to the bilayer interface, but buried deeply inside the hydrophobic region of the lipid bilayer.

Bacteriorhodopsin, BR, is a natural, photoresponsive, biomolecule that has potential application in data storage, imaging and sensing. Being membrane-bound, however, it is coupled with metallic electronic surfaces only with some difficulty. We report herein a facile method to generate uniformly orientated, anchored and active monolayers of BR on metallic electrodes. In the present study, the cytoplasmic side of the BR is equipped with an engineered cysteine to achieve largely lipid-free, orientation-specific, highly stable, covalent immobilization on gold surfaces. By using non-invasive Kelvin probe force microscopy, it is possible to measure the light-induced proton accumulation at the extracellular protein surface at truly molecular scales. The intimate probe-BR interaction possible on lipid removal facilitates the detection of photoinduced surface potential switching substantially larger ((20.4 ± 7.5) mV) with functional single delipidated mutant BR trimers than for the wild-type protein. The proton pumping detected is also notably highly unidirectional with the orientated protein.

Protein is the most important emulsion stabilizer from the Food Science point of view and its original structure and unfolding of the protein at the interface is of paramount importance for emulsion stability. Many different approaches have been taken to gathering more information about the emulsion phenomenon, but most of them result physical, interfacial or even empirical information about this process.'Molecular aspects of the lipid-protein interaction at the water-oil interface is scarce and often difficult to obtain. Microscopic, ...

1. Biochim Biophys Acta. 1998 Nov 10;1376(3):297-318. Solid-state NMR approaches for studying the interaction of peptides and proteins with membranes. Watts A. Biomembrane Structure Unit, Biochemistry Department, Oxford University, Oxford OX1 3QU, UK. awatts@bioch.ox.ac.uk. PMID: 9804977 [PubMed - indexed for MEDLINE]. Publication Types: Research Support, Non-US Gov't; Review. MeSH Terms. Animals; Cytochrome c Group/chemistry; ...

ABSTRACT Continental deformation is known to preferentially focus in young tectonic provinces, where the lithosphere has been weakened during previous subduction, rifting and orogenic events1-3. This suggests that pre-existing thermo-mechanical properties and composition of the crust and mantle lithosphere may control the structural styles that develop in collisional orogens. Although long-term crustal shortening is now fairly well quantified in a number of external zones of orogens, the reasons for the differences in crustal-scale deformation in these settings has rarely been addressed4,5. Here, we examine the relationship between long-term crustal shortening in collisional orogens that have developed on lithospheres of different thermo-tectonic age and proxies of long-term lithosphere strength. By analyzing more than 50 data sets from 30 fold and thrust belts, we show that shortening and thermotectonic age at time of shortening are correlated. In particular, deformation of Phanerozoic lithosphere, characterized by low elastic thickness (~20 km), high lithospheric temperature and fertile, weak, instable mantle is prone to involve mid-crustal detachment, thus reducing bulk crustal strain (<35%). Older cratonic blocks, characterized by higher elastic thickness (>60 km) and high-viscosity mantle promote stable underthrusting characterized by higher, more localized, shortening (up to ~70%) in weakly consolidated sediments. Our findings provide a key for linking distribution of continental deformation with secular cooling of the Earth and inherited properties of the lithosphere, including the nature of the sub-continental mantle and thermo-mechanical structure of the crust.

In this work we return to the possible uses of small-angle neutron scattering (SANS) in the study of single membrane structure in water solutions at low membrane concentrations (ie, in excess water).—Using the example of lipid vesicles the effectiveness of this method in the determination of membrane thickness will be shown and the limits within which this method can be used with the same aims will be set.—Most important is the fact that the use of SANS on single membranes in solution makes possible to determine the position of molecular ...

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1. Biochem Soc Trans. 1998 Aug;26(3):S297. Solid state NMR studies of ligands bound to the nicotinic acetylcholine receptor. Williamson PT, Watts J, Gröbner G, Miller KW, Watts A. Department of Biochemistry, University of Oxford, United Kingdom. PMID: 9766016 [PubMed - indexed for MEDLINE]. Publication Types: Research Support, Non-US Gov't. MeSH Terms. Acetylcholine/ analogs & derivatives; Acetylcholine/chemistry; Acetylcholine/metabolism ...

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The vertebrate rod outer segment (ROS) is in many ways an ideal system for spectroscopic and magnetic-resonance studies of lipid-protein and protein-protein interactions (Watts, 1982). The relative simplicity in composition and comparatively low metabolic activity in the ROS, coupled with the well characterized membrane and protein components, allows direct information to be deduced about the photoreceptor protein rhodopsin and its interaction with the neighbouring membrane lipids and soluble proteins. We have used relatively sensitive ...

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We demonstrate that (13)C-detected spectra recorded using fast (60 kHz) magic angle spinning on sub-milligram (<10 μmol) quantities of a protonated 7 trans-membrane helix protein (bacteriorhodopsin) in its native lipid environment are comparable in sensitivity and resolution to those recorded using 15-fold larger sample volumes with conventional solid state NMR methodology. We demonstrate the utility of proton-detected measurements which yield narrow (1)H linewidths under these conditions, and that no structural alterations are observed. We propose that these methods will prove useful to gain structural information on membrane proteins with poor availability, which can be studied in their native lipid environments.

Membrane proteins still represent a challenge to structural biologists, as evidenced throughout this volume. The statistics of the Protein Data Bank (PDB)[1] reflect the remarkable progress in structural biology; the number of structures solved every year has grown exponentially since the first two structures were deposited in 1972, and as of March 2007, there are 42 082 structures deposited. However, membrane proteins represent only a small fraction of the known structures (less than 2%), even though they constitute 20 to 40 ...

Solid state NMR is now established as a method for resolving structural information for large biomolecular complexes such as membrane-embedded proteins. In principle, there is no molecular weight limit to the use of the approach, although the complexity and volume of data is still outside complete assignment and structural determinations for any large (Mr>~ 40k) complex, unless specific methods are used to reduce the information content. Such methods include specific residue type labelling, labelling of putative segments of a protein ...

A pulsed EPR saturation-recovery method has been used to measure spin-lattice relaxation times (T 1,) for aminoxyl spin labels in cardiolipin bilayers, with and without cytochrome c. The relaxation time for each spin label was determined at various positions in the bilayer, which included the bilayer surface and three positions within the hydrophobic interior in the membrane, as well as a position close to the glycerol backbone of phospholipids in the bilayer. A dynamic profile for the hydrocarbon chains in bilayers was found in agreement ...

Abstract Knowledge of structural details at an atomic level is important in understanding the function and molecular properties of biomolecules. In situations where crystallography or solution state NMR methods are not applicable, solid state NMR can provide valuable insight without the limitations imposed by the availability of crystals, solubility or molecular weight. In particular dedicated solid state NMR methods enable the determination of selective internuclear distances at high accuracy. A popular and robust technique is ...

Archaerhodopsin 4 (AR4), a new member of the microbial rhodopsin family, is isolated from Halobacterium species xz515 in a Tibetan salt lake. AR4 functions as a proton pump similar to bacteriorhodopsin (BR) but with an opposite temporal order of proton uptake and release at neutral pH. However, further studies to elucidate the mechanism of the proton pump and photocycle of AR4 have been inhibited due to the difficulty of establishing a suitable system in which to express recombinant AR4 mutants. In this paper, we report a reliable method for expressing recombinant AR4 in Halobacterium salinarum L33 with a high yield of up to 20mg/l. Experimental results show that the recombinant AR4 retains the light-driven proton pump characteristics and photo-cycling kinetics, similar to that in the native membrane. The functional role of bacterioruberin in AR4 and the trimeric packing of AR4 in its native and recombinant forms are investigated through light-induced kinetic measurements, two-dimen...

The structural properties of isolated purified rat brain synaptosomal membranes, both in the presence and absence of purified active toxin of the Mojave snake Crotalus scutulatus scutulatus, were studied by spin-label electron spin resonance techniques. The spectra from eight different positional isomers of nitroxide-labelled stearic acids, a rigid steroid androstanol, and a spin-labelled phosphatidylcholine intercalated into the synaptosomal membranes, were obtained as a function of temperature from 4-40 degrees C. The flexibility gradient (from spin-label order parameters) and polarity profile (from isotropic splitting factors) across the synaptosomal membranes, was characteristic for lipid bilayers. The nitroxide spin-labelled steroid, androstanol, intercalated into the synaptosomal membrane, revealed the abrupt onset of rapid cooperative rotation about the long axis of the molecule at 12 degrees C showing that the lipid molecules are rotating rapidly around their long axes at physiological temperatures. The presence of the Mojave toxin affected the synaptosomal membrane in a complex manner, depending upon the temperature and the position of the nitroxide label on the alkyl chain of the stearic acid probe. Mojave toxin exerted little effect on the flexibility gradient of the synaptosomal membrane at 20 degrees C, a temperature at which the acyl chain labels detected a structural change in the membranes. At temperatures lower than 20 degrees C, the Mojave toxin produced a change in the flexibility gradient of the synaptosomal membrane which indicated an increased disordering in the upper region of the membrane and a concomitant increased ordering of the acyl chains in the deeper regions of the membrane. At temperatures higher than 20 degrees C, the order profile of the synaptosomal membrane was shifted by the presence of the Mojave toxin in a manner which indicated that the outer parts of the membrane were more rigid and the inner regions more fluid, than in controls. A cross-over point for the perturbation occurred at C8-9, which is about 12-14 A into the membrane. This is the approximate depth of the hydrophobic pocket shown in pancreatic phospholipase A2 [Drenth et al. (1976) Nature (Lond.) 264, 373-377], a protein likely to be homologous to the basic subunit of the toxin. At all temperatures, rotational lipid motion was inhibited by the toxin as indicated by the steroid probe. The electron spin-resonance spin-label results are interpreted in terms of the partial penetration of the basic subunit of the intact toxin into the membrane, disordering the ordered chains at low temperature and ordering the disordered chains at physiological temperatures. The purified individual toxin subunits did not perturb the membrane lipids at physiological temperatures implying that both subunits must be associated for activity of the toxin which is confirmed by toxicity studies.