Saturation transfer electron spin resonance of Ca2(+)-ATPase covalently spin-labeled with beta-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels.

The Ca2(+)-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide beta-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the alpha-carbon, to enhance further the electrophilic character of the beta-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2(+)-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the linewidths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer lineheight ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the lineheight ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the lineheight ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2(+)-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.     

Saturation transfer EPR measurements of the rotational motion of a strongly immobilized ouabain spin label on renal Na, K-ATPase

The rotational motion of an ouabain spin label with sheep kidney Na,K-ATPase has been measured by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) measurements. Spin-labelled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 ± 0.1 mol of bound ouabain spin label per ATPase β dimer. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (> 99%) of a broad resonance which is characteristic of a strongly immobilized spin label. ST-EPR measurements of the spin labelled ATPase preparations yield effective correlation times for the bound labels of 209 ± 11 μs at 0°C and 44 ± 4 μs at 20°C. These rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements with glutaraldehyde-crosslinked preparations indicated that the observed rotational correlation times predominantly represented the motion of entire Na,K-ATPase-containing membrane fragments, rather than the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The strong immobilization of the ouabain spin label will make it an effective paramagnetic probe of the extracellular surface of the Na,K-ATPase for a variety of NMR and EPR investigations.     

Spin label saturation transfer ESR studies of protein-lipid interactions in Photosystem II-enriched membranes

Saturation transfer ESR has been used to study the dynamic behaviour of lipids in the appressed regions of thylakoid membranes from pea seedlings. Four different phospho- and galacto-lipid spin labels (phosphatidylcholine labelled at the 12 or 14 C-atom positions of the sn-2 chain, phosphatidylglycerol labelled at the 14-position of the sn-2 chain, and monogalactosyldiacylglycerol labelled at the 12-position of the sn-2 chain) were used to probe the lipid environment in photosystem II-enriched membranes prepared by detergent extraction. The ESR spectra show that the majority of the lipid in these preparations is strongly motionally restricted. Values for the effective rotational correlation times of the labelled chains were deduced from the lineheight ratios and integrals of thhe saturation transfer ESR spectra. The effective rotational correlation times were found to be in the 10⁵ range, indicating a very low lipid chain mobility which correlates with the low lipid content of these preparations. Comparison of the effective rotational correlation times deduced from the different diagnostic regions of the spectrum revealed little anisotropy in the chain mobility, indicating that the dominant motional mode was trans-gauche isomerization. The effective rotational correlation times deduced from the spectral integrals were similar to those deduced from the lineheight ratios, consistent with the absence of any appreciable fluid lipid component in these preparations. The results also indicate some selectivity of interaction between the lipid species, with phosphatidylcholine exhibiting appreciably slower motion than either phosphatidylglycerol or monogalactosyldiacylglycerol.     

Molecular motion and order in oriented lipid multibilayer membranes evaluated by simulations of spin label ESR spectra. Effects of temperature,cholesterol and magnetic field

A simulation method to interpret electron spin resonance (ESR) of spin labelled amphiphilic molecules in oriented phosphatidylcholine multibilayers in terms of a restricted motional model is presented. Order and motion of the cholestane spin label (3-spiro-doxyl-5α-cholestane) incorporated into egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, pure and in mixture with cholesterol, were studied at various termperatures. With egg yolk phosphatidylcholine identical sets of motional parameters were obtained from simulations of ESR spectra obtained at three microwave frequencies (X-, K- and Q-band). With dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine analyses of the spectra show that phase transitions occur in samples containing up to 30 mol % cholesterol. The activation energy for the motion of the spin label is about three times larger above than below the phase transition, indicating a more collective motion in the liquid crystalline state than in the gel state. In the liquid crystalline state the activation energy is larger in the pure phosphatidylcholines than with cholesterol added. Additions of cholesterol to egg phosphatidylcholine induces a higher molecular order but does not appreciably affect correlation times. This is in contrast to dipalmitoylphosphatidylcholine where both order and correlation times are affected by the presence of cholesterol. The activation energies follow the same order as the transition temperatures: dipalmitoylphosphatidylcholine > dimyristoylphosphatidylcholine > egg yolk phosphatidylcholine, suggesting a similar order of the cooperativity of the motion of the lipid molecules. Magnetic field-induced effects on egg phosphatidylcholine multibilayers.     

A spin labelling study of immunomodulating peptidoglycan monomer and adamantyltripeptides entrapped into liposomes

The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo.     

Molecular motion and order in oriented lipid multibilayer membranes evaluated by simulations of spin label ESR spectra. Effects of temperature, cholesterol and magnetic field.

A simulation method to interpret electron spin resonance (ESR) of spin labelled amphiphilic molecules in oriented phosphatidylcholine multibilayers in terms of a restricted motional model is presented. Order and motion of the cholestane spin label (3-spiro-doxyl-5alpha-cholestane) incorporated into egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, pure and in mixture with cholesterol, were studied at various temperatures. With egg yolk phosphatidylcholine identical sets of motional parameters were obtained from simulations of ESR spectra obtained at three microwave frequencies (X-, K- and Q-band). With dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine analyses of the spectra show that phase transitions occur in samples containing up to 30 mol % cholesterol. The activation energy for the motion of the spin label is about three times larger above than below the phase transition, indicating a more collective motion in the lipid crystalline state than in the gel state. In the liquid crystalline state the activation energy is larger in the pure phosphatidylcholines than with cholesterol added. Additions of cholesterol to egg phosphatidylcholine induces a higher molecular order but does not appreciably affect correlation times. This is in contrast to dipalmitoylphosphatidylcholine where both order and correlation times are affected by the presence of cholesterol. The activation energies follow the same order as the transition temperatures: dipalmitoylphosphatidylcholine greater than dimyristoylphosphatidylcholine greater than egg yokd phosphatidylcholine, suggesting a similar order of the cooperativity of the motion of the lipid molecules. Magnetic field-induced effects on egg phosphatidylcholine multibilayers were found at Q-band measurements above 40 degrees C. The cholestane spin label mimics order and motion of cholesterol molecule incorporated into the lipid bilayers. This reflects order and motion of the portions of the lipid molecules on the same depth of the bilayer as the rigid steroid portions of the intercalated molecules.     

Electron spin resonance study of human transcortin binding properties. Interaction of monomeric and polymeric forms with spin labeled corticosteroids.

Corticosteroids bearing a nitroxide radical on the side chain were shown to bind with a high affinity to purified human serum transcortin. ESR spectroscopy data allowed calculation of the thermodynamic parameters (ΔH, ΔS) of the interaction and characterization of a hydrophobic spin label binding site. Transcortin spontaneously associated upon storage into reversible polymeric forms which partly retained the steroid binding properties. Apparent rotational correlation times of 34 and 70 n sec were obtained by ESR analysis at 20°C for the transcortin monomer and dimer respectively.     

Low microwave-amplitude ESR spectroscopy: measuring spin-relaxation interactions of moderately immobilized spin labels in proteins

Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1)<0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T(2e), is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-I252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T(2e). We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.     

Analysis of existing models of spin label movement during spin labeling of biological macromolecules

The spin-labeled bovine serum albumin and IgG were studied in search of an experimental approach for comparison of different models of rotational mobility of spin label. These models are: the model of isotropic motion of spin label together with the macromolecule (IM); the model of highly anisotropic motion of spin label (HAM); and the model of slow isotropic motion of label around the binding site (SIML). The experimental spectra were measured on a common X-band ESR spectrometer and on the unique 140 GHZ (lambda = 2 mm) ESR spectrometer under the same conditions. Theoretical spectra were computer-calculated according to Freed's theory. We have found, that the results of temperature-viscosity experiments in X-band are contradictory to the model of IM both for the BSA and IgG species. The models of HAM and SIML for the BSA give identical X-band spectra. The bovine serum albumin spectra in the 2 mm region strongly contradict to the assumptions of the HAM model. Also, the SIML model fails to describe the experimental spectra in terms of isotropic motion of the spin label around the binding site. X-band spectra of IgG can not be explained by the SIML model, while the same spectra in the 2 mm region can not be explained by the HAM model.     

Electron paramagnetic resonance study of the interactions between steroid hormones and binding proteins]

Interaction of a spin labeled corticosteroid (desoxycorticosterone nitroxyde: DOC -NO) with three purified proteins (albumin, transcortin, progesterone binding protein: PBG) was studied by electron spin resonance (ESR) spectroscopy. DOC-NO was competitive with natural corticosteroids and therefore bound at the same site to specific binding proteins. ESR spectra in the presence of each of the proteins showed an immobilized (bound) form of the spin labeled steroid and allowed the calculation of the corresponding association constant (Ka) at equilibrium. The three binding proteins could be characterized by the ESR parameters of the DOC-NO bound form. The thermodynamic parameters (deltaH, deltaS) of the steroid-protein interactions were calculated from the ESR data obtained within a wide temperature range (3--40 degrees C). The ESR spectra width (2T) was used to evaluate the polarity of the spin label environment within the steroid binding site: a hydrophobic character was observed for transcortin whereas PBG exhibited a more hydrophilic steroid binding sits. The rotational correlation time of the three protein DOC-NO complexes at equilibrium were calculated from ESR data; the results were correlated with the protein molecular size and suggested a non spherical shape for the binding macromolecule in solution. Spin labelling of biologically active steroids thus provides a novel approach for the study of the interaction of these hormones with their binding protein. Providing a suitable spin label, the ESR parameters may allow the characterization of several types of binding sites of different biological significance for the same hormone, in biological fluids as well as in target tissues.     

A spin label study of rat brain membranes. Effects of temperature and divalent cations.

Rat brain myelin, synaptosomal plasma membranes and synaptic vesicles were spin labelled with stearic acid nitroxide derivatives. Their electron spin resonance spectra were studied as a function of temperature and devalent ions (Ca2+ and Mg2+) concentrations. (1) Synaptosomal plasma membranes and synaptic vesicles show identical temperature variations of their order parameter (S = 0.58 at 35 degrees C and S = 0.72 AT 22 DEGREES C). Myelin appears more rigid (S = 0.66 at 35 degrees C and S = 0.76 at 22 degrees C). A discontinuity of the order parameter variation as a function of temperature, is observed between 14.5 degrees C and l9.5 degrees C with the three types of membranes. (2) The hydrophobic core of these membranes is very fluid. No transition temperature is observed. The measured values of the spin label rotation correlation times and rotational activation energies are 2.1 and 2.8 ns at 35 degrees C and 3.1 and 3.6 kcal/mol respectively for synaptosomal plasma membranes and myelin. (3) Ca2+ enhances the membrane rigidity (12+/-0.7% increase of the order parameter at 35 degrees C in the presence of 10(-3) M Ca2+) and increases the transition temperature. At a lower extend, similar effects are observed with Mg2+.     

Interaction of albumin with the endothelial cell surface

Endothelial cells (EC) are covered with cell-borne proteoglycans and glycoproteins. Blood plasma proteins (e.g., albumin) adsorb to this glycocalyx forming a complex endothelial surface layer (ESL). We determined the molecular mobility of albumin by electron spin resonance (ESR) in the presence and absence of ECs to analyze interactions with the ESL. Albumin was spin labeled with 5- or 12-4,4-dimethyloxazolidine-N-oxyl (DOXYL)-stearic acid yielding information on the mobility of the molecular surface (5-DOXYL) or the entire protein (12-DOXYL). EC cultures grown on glass coverslips were immersed in labeled albumin and placed in the temperature-regulated cavity of an ESR spectrometer. Alternatively, ECs were labeled and then exposed to native albumin. At 37 degrees C, rotational correlation times determined by modified saturation transfer ESR (ST-ESR) were 26 and 48 ns for 5-DOXYL- and 12-DOXYL-labeled albumin, respectively. Presence of ECs increased rotational correlation time values for 5-DOXYL-stearic acid to 37 ns but not for 12-DOXYL-stearic acid. Albumin was able to completely take up the label from labeled EC within 2 min. The present study shows that modified ST-ESR can be used to determine the mobility of biological macromolecules interacting with cellular surfaces. Reduction in albumin surface mobility in the presence of EC at unchanged mobility of protein proper and fast removal of labeled fatty acids from EC membranes indicate rapid transient interactions between albumin surface and ESL but no rigid incorporation of albumin into a macromolecular network that would interfere with its transport function for poorly water-soluble substances.     

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.     

Study on the interaction between hemoglobin Izu (Macaca) and organic phosphates by spin labeling

ESR spectra of the carbonmonoxy, oxy, and deoxy derivatives of hemoglobin Izu [Hb Izu (Macaca): beta 83 (EF 7) Gly leads to Cys] labeled at cysteine beta 83 with maleimide spin label have been observed in the presence and absence of 2,3-diphosphoglycerate and inositol hexaphosphate. The tau c values obtained from the spectra indicated that inositol hexaphosphate binds to all the derivatives of Hb Izu, but 2,3-diphosphoglycerate only to the deoxy derivatives.     

Transfer of cholestane spin label between lipid bilayer membranes and its molecular motion in membranes.

The relation between the molecular motion of a steroid in lipid membranes and the transfer rate between membranes was examined using radioactive cholestane spin label. Order parameters of the molecule were determined in bilayers composedof dipalmitoylglycerophosphocholine or egg yolk phosphatidylcholine at various temperatures. The line widths of the ESR signal of the cholestane spin label in membranes, which depend upon the rate of molecular axial rotation in the membranes, were also measured. The temperature dependences of these two parameters and of the transfer rate suggest a close correlation between the rate of molecular axial rotation and the transfer rate.     

Electron spin resonance spin label studies of plasma fibronectin: effect of temperature

Plasma fibronectin was chemically modified by 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (maleimide spin label). Only the free sulfhydryl groups of plasma fibronectin were modified by the label under the experimental conditions. The ESR spectrum of spin-labeled fibronectin showed that the sites of labeling were highly immobilized, suggesting that the sulfhydryl groups of the protein are in small, confined environments. The conversion of the strongly immobilized ESR spectrum into a weakly immobilized one was observed when the spin-labeled protein was heated from 30 to 60 degrees C, indicating the thermal unfolding of the protein molecules. The midpoint temperature for the thermal unfolding of plasma fibronectin is about 50 degrees C. The results suggest that plasma fibronectin is stable to about 40 degrees C and starts unfolding above this temperature. The rotational correlation time estimated from the ESR spectrum of spin-labeled fibronectin at 21 degrees C was about 2.0 X 10(-8) s. The rotational correlation time calculated from the Stokes-Einstein equation, assuming a rigid globular configuration for fibronectin with a Stokes radius of 10 nm, was about 7.8 X 10(-7) s. The differences in rotational correlation time by a factor of 39 between experimental and calculated values do not support a globular configuration for plasma fibronectin.     

Location and dynamics of alamethicin in unilamellar vesicles and thylakoids as model systems. A spin label study

Location and dynamics of the voltage-dependent pore-forming icosapeptide alamethicin have been studied using spin labels which were linked directly and via spacers to the C-terminus of the amphiphilic alpha-helix. Ion-transport activities of these derivatives were found to be very similar to those of natural alamethicin in green plant thylakoids chosen as a model system. The shape of the electron spin resonance spectra indicates segmental motion of the nitroxide rather than rotation of the whole peptide. A population of spins showing narrow lines in the presence of thylakoids or lipid vesicles is attributed to alamethicin in the aqueous solution. A second population shows rotational correlation times greater than 10(-9) s and is bound to the membranes, the C-termini residing in an environment with a polarity close to that of water. This population is inaccessible to the hydrophilic, charged line broadening agent chromium oxalate. Since spectral shapes and amplitudes of spectra are unchanged by additions of unlabelled peptide, it is concluded that the ESR detectable spins are bound to peptides essentially in the monomeric state. Alamethicin induced pore formation under flash illumination is demonstrated by measurement of kinetics of proton deposition in the thylakoid interior. When pores are opened by illuminating thylakoids and thus applying a membrane potential, mainly the bound population is affected by a process reversibly suppressing the signal, whereas only limited disappearance of label from the external medium is detected. Apparently, the potential causes a change in the conformation of the peptide which leads to a further immobilisation of the label, possibly due to a deeper insertion of the alpha-helices into the lipid membrane. However, evidence has been presented experimentally that there is no detectable change of potential prior to the opening of the pore.     

Spin-label techniques for monitoring macromolecular rotational motion: empirical calibration under nonideal conditions.

Practical techniques are demonstrated for determining rotational correlation times of macromolecules from the first harmonic absorption electron spin reasonance spectra of tightly bound spin labels. The techniques are developed to compensate for such nonideal conditions as residual label motion, temperature dependence of rigid limit spectral parameters, and the presence of inhomogeneous line broadening. These effects are all shown to be of importance in monitoring the rotational motion of carbonmonoxyhemoglobin which is spin labeled with the tightly bound nitroxide label, 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxy. Spin-label interactions with other paramagnetic agents are also shown to produce spectral changes which are qualitatively similar to, but quantitatively different from, those resulting from increases in the rate of rotational motion.     

Electron spin resonance study of human alpha 1-acid glycoprotein interaction with a spin labelled steroid.

The interaction of human alpha 1-acid glycoprotein (AAG) with a corticosteroid was studied using nitroxide labeled deoxycorticosterone and electron spin resonance (ESR) spectroscopy. The ESR spectra of the spin labeled steroid in the presence of AAG could be used to characterize the ligand-protein interaction at equilibrium without the need of a separation between bound and free species. An association constant Ka of 6.10(5) M-1 at 20 degrees C and a binding capacity of one site per mole protein were found. ESR spectra recorded at equilibrium at various temperatures allowed the calculation of enthalpy and entropy variations for the steroid-protein interaction; these thermodynamic parameters exhibited a rapid change above 45 degrees C which may be related to a protein conformational modification above this temperature, as detected by circular dichroism study. The ESR spectra width could be used to define a polar character for the spin label environment in the steroid binding site of AAG and to calculate an apparent rotational correlation time of 2.8 x 10(-8) sec for the steroid-protein complex in aqueous solution at 20 degrees C. It can be concluded that spin labeling and ESR methodology is of value in the study of steroid-protein interactions of biological significance above all because it can provide direct physico-chemical information concerning the local environment of the ligand in its binding site at equilibrium.     

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 $\text{g-}\text{values}$, 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.