Spin-labeled Molecules Research Articles

Analysis of the ESR spectra of the cholestane spin label in oriented multibilayers of lecithin and cholesterol in the gel state

Abstract The ESR spectra of the cholestane spin label in macroscopically oriented multibilayers of dipalmitoyllecithin and cholesterol have been studied in the temperature region from −17 to 40°C, below the liquid crystalline transition temperature of these systems. A qualitative analysis of the angular dependence of the powderlike ESR spectra is given, showing that in the pure lecithin system the molecules are tilted, whereas in multibilayers with 10 mole % cholesterol or more, no tilt of the molecules is found. The ESR spectra were computer simulated, using a simple exponential distribution function for the orientation of the spin label molecules in the bilayer, in terms of a static tilt angle and spread angle. In addition the components of the g -factor and hyperfine tensors of the cholestane spin label have been determined. A comparison has been made of the components of the g-factor and hyperfine tensors obtained from the computer simulation and components reported in the literature, which have been obtained with spin labels in single-crystal hosts.

An ESR study of the hydration steps of lecithin multilayers

A spin-labeled stearic acid and other spin-labeled molecules have been used for an ESR study of the hydration process of the egg lecithin lamellar phase for water concentrations, CW greater than 10%. Two hydration steps are found for CW approximately 20% and CW congruent to 30%, in good agreement with results of the literature. To explain discontinuities in the behavior of three spin-labeled molecules at CW congruent to 20%, a conformational change of the phosphatidylcholine polar head and a phase transition in the polar interface are proposed.

Cytochrome c induced lateral phase separation in a diphosphatidylglycerol-steroid spin-label model membrane

The extrinsic membrane protein cytochrome c binds to lipid mixtures containing negatively charged phospholipids such as diphosphatidylglycerol (DPG). In this study the effect of cytochrome c on the lipid distribution in a DPG-steroid spin-label (3-doxyl-5alpha-cholestane) model membrane system is examined. The electron spin resonance (ESR) line-shape changes indicate that cytochrome c induces lateral phase separation at room temperature. The resulting two-dimensional lipid distribution is nonrandom, consisting of clusters of phospholipids bound to cytochrome c and patches of steroid spin-label molecules. Phase separations are also observed in the three-component system: DPG, phosphatidylcholine, and 3-doxyl-5alpha-cholestane.

On the location of active serines of membrane acetylcholinesterase studied by the ESR method

1. An attempt was made to find out the causes of the discrepancy between the ESR spectra of membrane acetylcholinesterase (EC 3.1.1.7) obtained by Morrisett and co-workers and those obtained by the present authors. 2. In order to see whether the discrepancy was due to the different spin-labeling procedures, the same membrane acetylcholinesterase preparations were spin-labeled with the same compound, using the two different spin-labeling procedures. The enzyme activity was determined with pH-static titration and the ESR spectra recorded. 3. It was found that after spin-labeling according to Morrisett and co-workers, there were from 10-100 times more spin-label molecules bound to the enzyme preparations than there were active serines in them. 4. Using the method of Morrisett and co-workers, the majority of spin-label molecules was found to be bound to sites outside the active serines whereas the spin-labeling procedures of the present authors proved to be selective for active serines; the discrepancy in ESR spectra is explained.

Changes in the restriction of molecular rotational diffusion of water-soluble spin labels during fatty acid starvation of yeast

Yeast mutants lacking fatty acid synthetase activity (fas-) die when deprived of saturated fatty acid under conditions which are otherwise growth-supporting. The spin label technique is used to show that restriction of molecular rotational diffusion of spin label molecules dissolved in aqueous zones increases several fold under conditions of fatty acid starvation while the apparent physical state of cellular hydrocarbon zones remains essentially unchanged. We focus attention on the cellular aqueous interior as the potential site of alteration under selective starvation conditions. Correspondences exist between restriction of molecular motion of water soluble spin labels dissolved in the cell and loss of cell viability. The correspondences to changes in the molecular motion of hydrocarbon soluble spin labels are much less or are not detectable.

Protein—surfactant interactions. Spin label study of interactions between bovine serum albumin (BSA) and sodium dodecyl sulphate (SDS)

Measurements have been made of changes in the mobility of spin-labelled stearic acid molecules bound at the high affinity sites of BSA as increasing numbers of SDS molecules become bound. Evidence is obtained for an increase in mobility with increasing SDS binding numbers (n) until n > 180 when all the stearic acid molecules are displaced from the protein. The results are explained in terms of (i) destruction of the high affinity binding sites when more than 10 SDS molecules become bound, and (ii) association of SDS molecules on the protein. On succinylation of BSA there is an increase in protein chain mobility resulting from unfolding of the protein molecule and this similarly leads to loss of the high affinity sites.

Paramagnetic spin label interactions with the envelope of a group A arbovirus. Lipid organization

Electron paramagnetic resonance observations were made on nitroxide spin- labeled molecules which were bound to the TC-83 vaccine strain of Venezuelan equine-encephalomyelitis virus. Paramagnetic resonance parameters derived from the observations and their dependence on sample temperature were similar but not identical to those which have been reported for these labels dissolved in lipid bilayer membranes of mammalian and bacterial origin. The data has a mechanical rigidity substantially greater than that of bilayers in cellular membranes. A model is presented which assumes the location of the lipid bilayer outside the nucleoprotein capsid and inside a spherical layer of envelope proteins. The model is in accord with Harrison's X-ray diffraction results for Sindbis virus. The model is discussed in terms of its implications with respects to the role played by lipid in viral maturation and infectivity.

ESR spectral analysis of the molecular motion of spin labels in lipid bilayers and membranes based on a model in terms of two angular motional parameters and rotational correlation times

Electron spin resonance (ESR) spectral line shapes are calculated for a nitroxide spin-labeled molecule undergoing rapid restricted rotations (twisting) about its long molecular axis while simultaneously tumbling within a cone. Explicit expressions are derived for the hyperfine splittings and g-values, as well as for the secular contributions to the motionally modulated linewidths. The present model is useful for analyzing the restricted twisting and tumbling motions, and rotational correlation times, of spin-labeled molecules in bilayers. Simulated spectra compare well with experimental spectra of lecithin bilayers marked with cholestane spin label, over a wide temperature range.

Pseudomonas putida cytochrome P-450. Binding of a spin-labeled analog of the inhibitor metyrapone

A spin-labeled analog of metyrapone, 1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl nicotinate, is similar to metyrapone in its effect on the absorbance spectrum of camphor-free ferric cytochrome P-450, shifting the Soret band from 418 to 420 nm. Spectrophotometric studies demonstrate that binding of the spin-labeled molecule is competitive with substrate (camphor) binding and yield a value of 4.1×105 m −1 for the equilibrium association constant Ka of the spin label-enzyme complex. Addition of cytochrome P-450 to an aqueous solution of the spin label considerably diminishes the EPR signal of the free, or unbound, spin label, which can be restored to a large extent by subsequent addition of camphor or metyrapone. The value of Ka determined from EPR titrations is consistent with that cited above. The EPR signal of the spin label bound to cytochrome P-450 is that of an immobilized nitroxide species in proximity to the paramagnetic ferric cation of the hemeprotein. From EPR data, the distance between these two paramagnetic species bound to the same enzyme molecule has been determined. The experimental results indicate that the spin label is directly coordinated through its pyridine nitrogen atom to the heme iron of cytochrome P-450 and that metyrapone binds similarly.

Electron spin resonance studies of spin-labeled mammalian cells by detection of surface-membrane signals.

Lipid-soluble spin labels were incorporated into human lymphocytes and mouse L-cells and the resulting electron spin resonance spectra were compared with spectra obtained from similarly labeled human erythrocytes. Spin labels were found in all subcellular fractions of the nucleated cells that contained membranes. Spinlabeled cells remained viable and capable of replicating in vitro. Electron spin resonance signals from spin-labeled nucleated cells underwent a time- and temperature-dependent decay that was reversed by bathing the cells in K(3)Fe(CN)(6). The demonstration of a relative cell impermeability to ferricyanide, as measured by both colorimetric and radioisotopic label methods, indicated that only spin-labeled molecules in the surface membrane were reactivated when ferricyanide was added to spin-labeled cells after the electron spin resonance signal had decayed.

Magnetic resonance in ordered lecithin-cholesterol multilayers

Abstract Lecithin-cholesterol multilayers, oriented between glass plates, have been studied with ESR of spin labels and with NMR. From the ESR spectra of the cholestan nitroxide label the order parameters have been derived as a function of temperature and degree of hydration. The rotational correlation times and activation energies for the anisotropic motion of the spin label molecules were determined at different degrees of hydration. From the NMR measurements strong evidence is obtained that the cholesterol and alkyl chains of lecithin are well oriented and are in rapid anisotropic motion.

Spin-labelled lipid-protein complexes

1. A study of the isooctane-soluble complexes formed between mixtures of phospholipids and some basic proteins has been made using the technique of spin-labelling. 2. The ESR spectra of the spin-labelled proteins (cytochrome c, lysozyme, histone, protamine and poly-l-lysine) were observed for aqueous solutions of the proteins, aqueous dispersions of the lipid-protein complexes and isooctane solutions of the same complexes. 3. An ionic combination of phospholipids and basic proteins was directly confirmed by the results. 4. The freedom of motion of the spin-label molecules with respect to the protein depends on the hydrophobic property of the phospholipid chains, which, because they are not directly involved in the complex formation, take up a conformation dependent upon the solvent. 5. Calculation of the reorientation correlation times of the labelled complexes in isooctane suggests the predominant existence of a complex formed between an individual protein molecule and several phospholipids.