The use of a maleimido spin label to detect conformational changes in cytochrome c oxidase.

Spin labeling with a maleimido spin label has been used to investigate conformational changes of bovine cytochrome c oxidase. These experiments show that the spin label is immobilized to a lesser degree when the enzyme is in the “oxygenated” form than it is in the oxidized state and support the view that the oxygenated form is a conformational variant. Experiments in which the maleimido spin-labeled cytochrome c oxidase was titrated with H₂O₂ reveal that the peroxide-treated enzyme, although possessing an absorption spectrum similar to that of the oxygenated form, has an electron paramagnetic resonance (epr) spectrum that is different from that of either the oxygenated form or the oxidized state. Extremes of pH cause a marked decrease in the degree of immobilization of maleimido spin labels bound to the oxidase. Alterations in the epr spectrum are reversible if the pH is held between 5.3 and 10.2 but are irreversible outside that range. Urea and guanidine hydrochloride also decrease the immobilization of the spin labels bound to the oxidase. The nature of the epr spectra indicates that under these conditions the enzyme assumes a more open conformation. Exposure to concentrations of sodium dodecyl sulfate as high as 10% does not result in as much loss of the immobilization as with urea or guanidine. Detergents such as cholate, Tween 80, and Triton X-100 have no significant effect on the epr spectrum of maleimido spin-labeled cytochrome c oxidase.     

The contact sites of sickle hemoglobin as seen by spin probe-spin label techniques

The spin-labeled tryptophan (TrpSL) was used as a structural probe of hemoglobin (Hb) contact sites. The electron paramagnetic resonance spectral data indicated that the probe exhibits weak binding to Hb with a dissociation constant of 3.2 x 10(-5) and 4.0 mol bound per Hb tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.5 s. The topology of the contact sites was investigated by using a dual spin label methodology in which TrpSL and 2H-15N covalently bound to B 93 cysteine residue were used. The electron spin resonance spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled Hb. The environment of the contact sites is discussed.     

The molecular dynamics of actin measured by a spin probe attached to lysine

Rabbit skeletal muscle G-actin was labeled with a spin probe, 3-(5-fluoro-2,4-dinitroanilino)proxyl. Tryptic digestion of the labeled actin followed by ultrafiltration and ion-exchange column chromatography indicated that the label was attached to residue Lys-61. This residue is found within a 9-kDa N-terminal segment that is easily degraded by proteolytic enzymes. The rate of reduction of the nitroxide bond by ascorbate was measured to determine the accessibility of the probe to small molecules in the solvent. These experiments showed that label bound to G-actin was relatively inaccessible to ascorbate, suggesting that it is buried within the protein structure. Polymerization further decreased the accessibility of the probe. Replacing bound Ca2+ with Mn2+ decreased the observed intensity of the electron paramagnetic resonance signal, indicating the spin label is about 2 nm distant from the metal binding site on the actin molecule. Labels attached to G-actin displayed an absorption spectrum characteristic of rotational motion with a correlation time (tau c) of 7 X 10(-9) s, which is faster than that for the whole molecule. Labels attached to F-actin had a value of tau c, measured using saturation transfer electron paramagnetic resonance, of 2 X 10(-5) s, which shows that the probe has a greater degree of mobility than the filament. The binding of heavy meromyosin or troponin-tropomyosin to labeled actin resulted in a further increase in the rotational correlation times, with the greatest decrease in mobility (tau c = 1 X 10(-4) s) observed when both were bound. Together the above results suggest that the 9-kDa segment of actin is mobile relative to the rest of the molecule and that this mobility can be influenced by the binding of heavymeromyosin or troponin-tropomyosin.     

A new bifunctional spin-label suitable for saturation-transfer EPR studies of protein rotational motion

A new bifunctional spin-label (BSL) has been synthesized that can be immobilized on the surface of proteins, allowing measurement of rotational motion of proteins by saturation-transfer electron paramagnetic resonance (STEPR). The spin-label contains a photoactivatable azido moiety, a cleavable disulfide, and a nitroxide spin with restricted mobility relative to the rest of the label. The label reacts with surface lysine residues modified with beta-mercaptopropionate. Bifunctional attachment is achieved by photoactivation of the azido group. Any spin-label that remains monofunctionally attached after photolysis is removed by reduction of the disulfide. Only bifunctionally attached BSL remains on the protein. Hemoglobin was used to test the utility of the BSL in STEPR by comparison with hemoglobin modified with maleimide spin-label (MSL), a commonly used standard for the STEPR technique. MSL is a monofunctional spin-label which is fortuitously immobilized by local protein structure within hemoglobin. The BSL labeling of hemoglobin did not significantly affect the quaternary structure of hemoglobin as determined by gel filtration chromatography. The conventional EPR spectra of the mono- and bifunctionally attached BSL-hemoglobin were similar to the MSL-hemoglobin spectrum, indicating that both forms of BSL were rigidly bound to hemoglobin. In contrast, the spectrum obtained by reaction of modified hemoglobin lysine residues with MSL indicated that these labels were highly mobile. The monofunctionally attached BSL was mobilized upon octyl glucoside addition whereas bifunctionally attached BSL was only slightly mobilized, suggesting that hydrophobic interactions immobilize the monofunctionally attached label on hemoglobin. The response of STEPR spectra of mono- and bifunctionally attached BSL-hemoglobin to changes in hemoglobin rotational correlation time was similar to the MSL-hemoglobin over the range of 10(-5)-10(-3) s. The spectra of bifunctionally attached BSL indicated slightly less motion than corresponding spectra for MSL or monofunctionally attached BSL. The new BSL is a good reporter of protein rotation and does not require unique protein structures for its immobilization on the protein. Thus, the BSL should be more generally applicable for STEPR studies of membrane protein rotation than existing monofunctional spin-labels.     

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.     

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.     

Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR.

The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled 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 mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, 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 of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.     

Evidence that the two free sulfhydryl groups of plasma fibronectin are in different local environments. Saturation-recovery electron spin resonance study.

Human plasma fibronectin is a dimer consisting of two subunits; each contains two cryptic thiol groups that were selectively labeled with an 15N,2H-maleimide spin label. Previous studies using conventional X-band electron spin resonance (ESR) methods showed that the spectrum of the labeled protein displays a single strongly immobilized component with an effective rotational correlation time of approximately 17 ns, suggesting that the physical environments of the two labeled sites per chain are indistinguishable. Here we have used saturation-recovery ESR to measure directly electron spin-lattice relaxation time (T1) of the labeled protein in solution at 27 degrees C. Interestingly, the time evolution of the signal was found to be biphasic, which was deconvoluted into two T1 values of 1.37 and 4.53 microseconds. Thus, the two spin-labeled sulfhydryl sites of plasma fibronectin (Fn), being similar in rates of rotational diffusion, differ by a factor of 3.2 in T1. Parallel experiments using various fibronectin fragments showed that the 1.37-microseconds component is associated with the label attached onto the thiol located in between the DNA-binding and the cell-binding domains, and the 4.53-microseconds component is associated with the label attached onto the thiol located within the carboxyl-terminal fibrin-binding domain. The data suggest that the saturation-recovery ESR is a useful method for differentiating multiple spin-labeled sites on macromolecules in which the labels undergo similar rates of rotational motion.     

Spin-label studies of the pH-induced random coil to α-helix conformational transition in poly(L-lysine)

Poly(L -lysine) of various molecular weights between 2700 and 475,000 was spin-labeled. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined by calculation of the rotational correlation time as the poly(L -lysine) underwent the pH-induced random coil to α-helix conformational transition. In general, the rotational correlation time of the nitroxide increased as the pH was increased, indicating a more restricted environment for the spin label when poly(L -lysine) is deprotonated. For the high-molecular-weight poly(L -lysine) this corresponds to the formation of the α-helix and indicates that the side chain–side chain interaction and decreased segmental motion of the backbone (slightly) restricts the motion of the spin label. For the 2700-molecular-weight poly(L -lysine), previously shown not to assume a helical conformation at high pH, the increase in the rotational correlation time of the spin label indicates that the side chain–side chain interaction takes place after deprotonation but without helix formation. This may indicate that helix formation per se is not needed to produce the observed effect even with the high-molecular-weight polymers. The rotational correlation time of the spin label at a particular pH did not depend on the molecular weight of the poly(L -lysine) over the 200-fold range of molecular weights. This indicates that the rotational correlation time reflects the rotational mobility of the spin label in a localized environment and not the rotational diffusion of the entire macromolecule.     

Preparation and properties of vesicles of a purified myelin hydrophobic protein and phospholipid a spin label study

Lipophilin, a hydrophobic protein purified from the proteolipid of normal human brain myelin, was recombined with phosphatidylcholine by solubilization of the lipid and protein in 2-chloro-ethanol followed by dialysis against buffer. This method resulted in homogeneous incorporation of the protein into lipid vesicles as judged by sedimentation on a sucrose gradient and freeze fracture electron microscopy. The lipid-protein vesicles were single layered, 1000–2000 Å in diameter and the freeze fracture faces contained intramembrane particles. The effect of lipophilin on the organization of the lipid was studied by use of spin label probes. Two distinct components were present in the spectrum of fatty acid spin labels in the lipid-protein vesicles. One was immobilized presumably due to the presence of boundary lipid around the protein and the second component was indicative of aniostropic motion similar to the spectrum in phosphatidylcholine vesicles and probably due to a lamellar phase but with a slightly greater order parameter. Lipophilin was found to increase the order parameter linearly with increasing concentration of protein incorporated into the vesicles. However, the phase transition temperature as measured from the 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) solubility parameter was unchanged.     

Stoichiometry, selectivity, and exchange dynamics of lipid-protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance. Effects of protein secondary structure.

Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis. Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation [cf. Spruijt, R. B., Wolfs, C. J. A. M., & Hemminga, M. A. (1989) Biochemistry 28, 9158-9165]. The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously [Wolfs, C. J. A. M., Horváth, L. I., Marsh, D., Watts, A., & Hemminga, M. A. (1989) Biochemistry 28, 9995-10001]. Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol. The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants. Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which labeled LC2 had been exchanged directly. However, the fraction of disordered probes was greater than seen in fibers. In summary, the above data indicate that the region of the myosin head proximal to the thick filament is ordered in rigor, and disordered in relaxation.     

Melting studies on spin-labeled polyadenylic–polyuridylic complexes

Temperature-dependent conformational transitions of spin-labeled poly rA, spin-labeled poly rU and the two-stranded helical complexes consisting either of spin-labeled rA·poly rU or spin-labeled poly rU·poly rA have been measured by electron spin resonance spectrocopy. The polynucleotides were spin labeled with 4-(2-iodoacetamido)2,2,6,6-tetramethylpiperidinooxyl and the spin label to nucleotide base ratio was approximately 1:600. The relationship between the log of tumbling time τ and the reciprocal absolute temperature for the spin-labeled single and double-stranded polynucleotides is presented. An agreement between T_m^OD (optical density melting) and T_m^sp (spin melting) is found for the complexes, which strongly supports the conclusion that the same temperature-dependent structural changes are monitored with both techniques.     

Saturation transfer electron paramagnetic resonance spectroscopy of spin labeled tobacco mosaic virus protein.

The coat protein of Tobacco Mosaic Virus is covalently labeled with a maleimide spin label at the single SH-group of the protein. Saturation transfer electron paramagnetic resonance spectroscopy, a technique that is sensitive to very slow molecular motion with rotational correlation times τ_c in the range 10^?7 to 10^?3 sec, shows the dissociation of large oligomers of spin labeled protein with τ_c～10^?4 sec at pH 5.5 to smaller oligomers at higher pH.     

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.     

The Role of the Fast Motion of the Spin Label in the Interpretation of EPR Spectra for Spin-Labeled Macromolecules

Abstract

The spin label method was used to observe the nature of the fast motions of side chains in protein monocrystals. The EPR spectra of spin-labeled lysozyme monocrystals (with different orientations of the tetragonal protein crystal in relation to the direction of the magnetic field) were interpreted using the method of molecular dynamics (MD). Within the proposed simple model, MD calculations of the spin label motion trajectories are performed in a reasonable real time. The model regards the protein molecule as frozen as a whole and the spin labeled amino acid residue as unfrozen. To calculate the trajectories in vacuum, a model of spin-labeled lysozyme was assembled, and the parameters of the force fields were specified for atoms of the protein molecule, including the spin label. The calculations show that the protein environment sterically limits the area of the possible angular reorientations for the NO reporter group of the nitroxide (within the spin label), and this, in turn, affects the shape of the EPR spectrum. However, it turned out that the spread in the positions of the reporter group in the angle space strictly adheres to the Gaussian distribution. Using the coordinates of the spin label atoms obtained by the MD method within a selected time range and considering the distribution of the spin label states over the ensemble of spin-labeled macro- molecules in a crystal, the EPR spectra of spin-labeled lysozyme monocrystals were simulated. The resultant theoretical EPR spectra appeared to be similar to experimental ones.     

Study of lysozyme by a spin-label method in the 2-mm range

Two millimeter range ESR-spectroscopy was used to measure the values of magnetic resonance parameters of egg lysozyme samples modified with nitroxyl label 4-(iodine-acetamide)-2,2,6,6-tetramethylpiperidine-1-oxyl by hist-15 residue. It has been shown that in a lyophilic sample the spin label forms a hydrogen bond with the protein group and when the sample is moistened--with water molecules. Temperature changes of the pattern of ESR line are analysed. It is concluded that in the moistened samples within 230-320 K the nitroxyl fragment of the label gets engaged in anisotropic movement with preferable rotation around Z-axis of N-O. fragment with anisotropy coefficient 5 and mean correlation time tau congruent to 5 X 10(-7) divided by 5 X 10(-8) c.     

Microscopic versus macroscopic diffusion in model membranes by electron spin resonance spectral-spatial imaging.

The macroscopic and the microscopic diffusion coefficients of a phospholipid spin label (16-PC) in the model membrane 1-palmitoyl-2-oleoyl-sn-glycero-phosphatidylcholine have been measured simultaneously in the same sample utilizing the new technique of spectral-spatial electron spin resonance imaging. The macroscopic diffusion coefficient Dmacro for self-diffusion of 16-PC spin label is obtained from imaging the concentration profiles as a function of time, and it is (2.3 +/- 0.4) x 10(-8) cm2/s at 22 degrees C. The microscopic diffusion coefficient Dmicro for relative diffusion of the spin probes is obtained from the variation of the spectral line broadening with spin label concentration, which is due to spin-spin interactions. Dmicro is found to be substantially greater than Dmacro for the same sample at the same conditions, and is estimated to be at least (1.0 +/- 0.4) x 10(-7) cm2/s. Possible sources for their difference are briefly discussed in terms of the models used for Dmicro.     

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.