High-field EPR and ESEEM investigation of the nitrogen quadrupole interaction of nitroxide spin labels in disordered solids: toward differentiation between polarity and proticity matrix effects on protein function

The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.     

Determining the topology of integral membrane peptides using EPR spectroscopy

This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC), was attached to the pore-lining transmembrane domain (M2delta) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14 degrees was calculated for the M2delta peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000-fold more sensitive than 15N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 microg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.     

A general approach for prediction of motional EPR spectra from Molecular Dynamics (MD) simulations: application to spin labelled protein

A general approach for the prediction of EPR spectra directly and completely from single dynamical trajectories generated from Molecular Dynamics (MD) simulations is described. The approach is applicable to an arbitrary system of electron and nuclear spins described by a general form of the spin-Hamiltonian for the entire motional range. It is shown that for a reliable simulation of motional EPR spectra only a single truncated dynamical trajectory generated until the point when correlation functions of rotational dynamics are completely relaxed is required. The simulation algorithm is based on a combination of the propagation of the spin density matrix in the Liouville space for this initial time interval and the use of well defined parameters calculated entirely from the dynamical trajectory for prediction of the evolution of the spin density matrix at longer times. A new approach is illustrated with the application to a nitroxide spin label MTSL attached to the protein sperm whale myoglobin. It is shown that simulation of the EPR spectrum, which is in excellent agreement with experiment, can be achieved from a single MD trajectory. Calculations reveal the complex nature of the dynamics of a spin label which is a superposition of the fast librational motions within dihedral states, of slow rotameric dynamics among different conformational states of the nitroxide tether and of the slow rotational diffusion of the protein itself. The significance of the slow rotameric dynamics of the nitroxide tether on the overall shape of the EPR spectrum is analysed and discussed.     

Low operational stability of enzymes in dry organic solvents: changes in the active site might affect catalysis

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme's initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.     

Oxidation of annelated diarylamines: analysis of reaction pathways to nitroxide diradical and spirocyclic products

Oxidation of diaryldiamine 2, a tetrahydrodiazapentacene derivative, provides diarylnitroxide diradical 1 accompanied by an intermediate nitroxide monoradical and a multitude of isolable diamagnetic products. DFT-computed tensors for EPR spectra and paramagnetic (1)H NMR isotropic shifts for nitroxide diradical 1 show good agreement with the experimental EPR spectra in rigid matrices and paramagnetic (1)H NMR spectra in solution, respectively. Examination of the diamagnetic products elucidates their formation via distinct pathways involving C-O bond-forming reactions, including Baeyer-Villiger-type oxidations. An unusual diiminoketone structure and two spirocyclic structures of the predominant diamagnetic products are confirmed by either X-ray crystallography or correlations between DFT-computed and experimental spectroscopic data such as (1)H, (13)C, and (15)N NMR chemical shifts and electronic absorption spectra.     

An EPR and NMR study of supramolecular effects on paramagnetic interaction between a nitroxide incarcerated within a nanocapsule with a nitroxide in bulk aqueous media

A 15N-labeled nitroxide was incarcerated into an octa acid nanocapsule, which was confirmed by 1H NMR and EPR spectroscopy. Electron paramagnetic interaction between the 15N-labeled incarcerated nitroxide and a 14N-labeled free nitroxide in the external aqueous solution was observed by EPR spectroscopy. The observation of spin-spin interaction, through the walls of the cancer and is reflected in the simultaneous line-broadening of both the 15N-labeled and 14N-labeled nitroxides. The computer-assisted analysis of the EPR data further provides direct information on the motion and the polarity of both the incarcerated paramagnetic nitroxide and the nitroxides in the external bulk aqueous phase. We also show how communication between an incarcerated guest and molecules in the bulk solvent can be enhanced or inhibited by supramolecular factors such as Coulombic attraction or repulsion between a charged guest@host complex (incarcerated 15N nitroxide) and charged molecules in the aqueous phase.     

EPR monitoring of sol-gel processes

Electron Paramagnetic Resonance (EPR) spectra of paramagnetic probes (free radical, 4-oxo-Tempo for non-doped glasses and vanadyl sulfate for doped silica glass) provide a non-intrusive means of monitoring of the dynamics of the sol-gel process. The free radical spectra parameters (rotation correlation time, isotropic hyperfine interaction parameter and line width) reflect changes in viscosity and polarity in the vicinity of the spin label. The EPR spectra of vanadyl can be used for monitoring the sol-gel process in that different types of mixed vanadyl complexes are formed during glass molding.     

Conformationally Restricted Isoindoline‐Derived Spin Labels in Duplex DNA: Distances and Rotational Flexibility by Pulsed Electron–Electron Double Resonance Spectroscopy

Three structurally related isoindoline‐derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation‐dependent pulsed electron–electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ^ExIm U , was synthesized and incorporated into DNA oligonucleotides. ^ExIm U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N?O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline‐based spin label to a uridine base. Continuous‐wave (CW) EPR measurements of ^ExIm U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ^Im U , which has restricted mobility due to an intramolecular hydrogen bond. The X‐band CW‐EPR spectra of ^ExIm U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ^Im U , ^Ox U , and ^ExIm U in duplex DNA showed a strong angular dependence for ^Im U , a medium dependence for ^Ox U , and no orientation effect for ^ExIm U . Thus, precise distances can be extracted from ^ExIm U without having to take orientational effects into account.     

New synthesis of a spin-labeled peptide nucleic acid and its interactions with nucleic acids

A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.     

Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy

We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)). Four single-site N(TAIL) mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N(TAIL) undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N(TAIL) and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N(TAIL) undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.     

Stearic acid spin labels in lipid bilayers: insight through atomistic simulations

Spin-labeled stearic acid species are commonly used for electron paramagnetic resonance (EPR) studies of cell membranes to investigate phase transitions, fluidity, and other physical properties. In this paper, we use large-scale molecular dynamics simulations to investigate the position and behavior of nitroxide spin labels attached to stearic acid molecules in dipalmitoylphosphatidylcholine (DPPC) bilayers. The results of these studies are potentially very important for the interpretation of EPR spectra, which rely on assumptions about the position of the label in the membrane. Additionally, we investigate the effect of chirality and ionization of the carboxyl group of the label. For a non-ionized species, we observe that spin-label molecules are even able to make flip-flop transitions between the leaflets of the bilayer. Such transitions have been previously observed only in very rare cases in molecular simulations.     

EPR analysis and DFT computations of a series of polynitroxides

Polynitroxides with varying numbers of nitroxide groups (one to four) derived from different aromatic core structures show intramolecular electron spin-spin coupling. The scope of this study is to establish an easy methodology for extracting structural, dynamical, and thermodynamical information from the EPR spectra of these polynitroxides which might find use as spin probes in complex systems, such as biological and host/guest systems, and as polarizing agents in dynamic nuclear polarization (DNP) applications. Density functional theory (DFT) calculations at the B3LYP/6-31G(d) level provided information on the structural details such as bond lengths and angles in the gas phase, which were compared with the single crystal X-ray diffraction data in the solid state. Polarizable continuum model (PCM) calculations were performed to account for solvent influences. The electron paramagnetic resonance (EPR) spectra of the polynitroxides in chloroform were analyzed in detail to extract information such as the percentages of different conformers, hyperfine coupling constants a, and rotational correlation times τ(c). The temperature dependence on the line shape of the EPR spectra gave thermodynamic parameters ΔH and ΔS for the conformational transitions. These parameters were found to depend on the number and relative positions of the nitroxide and other polar groups.     

EPR spectroscopic studies of the reduction of chromium(VI) by methanol in the presence of peptides. Formation of long-lived chromium(V) peptide complexes

The synthesis and characterization of the first Cr(V) complexes with non-sulfur-containing peptides, which may mimic the chemistry of the intermediates in the formation of Cr-induced peptide-DNA cross-links in vivo, are reported. The reduction of Cr(VI) with methanol in the presence of a number of non-sulfur-containing peptides produced relatively stable Cr(V)-peptide complexes, which were characterized by EPR spectroscopy and electrospray mass spectrometry. The reaction of Cr(VI) with methanol alone (in the absence of peptide ligands) resulted in the formation of two Cr(V)-methanol intermediates, with giso values of 1.9765 and 1.9687. The methanol reduction of Cr(VI) in the presence of the glycine peptides, triglycine, tetraglycine, and pentaglycine resulted in the formation of both Cr(V)-methanol and Cr(V)-peptide intermediates, while only the Cr(V)-peptide complexes were detected in the reactions with the alanine peptides trialanine, tetraalanine, and pentaalanine. Similar EPR signals were observed for all of the Cr(V)-peptide complexes with giso values between approximately 1.986 and approximately 1.979, and AN values of (2.1-2.6) x 10(-4) cm-1.     

EPR characterization of cellulose triacetate fibers used for enzyme immobilization

EPR studies of a nitroxide spin label and of the nitroxide spin-labeled albumin entrapped in cellulose triacetate fibers were carried out. The EPR spectra have shown that within the fiber only two phases are present: a liquid one of medium viscosity trapped inside microcavities, and a polymeric one surrounding them. After entrapment, spin-labeled albumin is distributed mainly in the liquid phase, though a not negligible amount of it remains within the polymeric matrix. The EPR studies have shown that, after the standard procedure of drying, the albumin is almost completely precipitated, but about 85% of it returns to solution when the fiber is again placed in the solution. The behavior of the albumin dissolved inside the microcavities toward denaturating agents and pH change, and that of the free albumin in solution is similar; the minor differences noticed indicate a second-order interaction between the fiber and the protein.     

Study on the heteroatom influence in pyridine-based nitronyl nitroxide biradicals with phenylethynyl spacers on the molecular ground state

Novel pyridine-based nitronyl nitroxide (NIT) biradicals, 3,5-bis[4-(1-oxyl-3-oxo-4,4,5,5-tetramethylimidazolin-2-yl)phenylethynyl)]pyridine (1) and 2,6-bis[4-(1-oxyl-3-oxo-4,4,5,5-tetramethylimidazolin-2-yl)phenylethynyl)]pyridine (2), and monoradicals, 4-(5-bromopyridine-3-ylethynyl)-1-(1-oxyl-3-oxo-4,4,5,5-tetramethylimidazolin-2-yl)benzene (3), 4-trimethylsilylethynyl-1-(1-oxyl-3-oxo-4,4,5,5-tetramethylimidazolin-2-yl)benzene (4), and 4-trimethylsilylethynyl-1-(1-oxyl-3-oxo-4,4,5,5-tetramethylimidazolin-2-yl)pyridine (5), were synthesized and investigated by ESR and UV-vis spectroscopy. The solution EPR measurements of the biradicals gave well-resolved, nine-line spectra with exact half line spacing as compared to monoradicals (giso = 2.0067) with isotropic line spacing /aN/= 7.36 G. This indicates strong, intramolecular exchange coupling (J > 7 x 10(-4) cm(-1); J/aN > 1) of the biradicals with in the limit of EPR. The temperature dependence on the Deltams = +/-2 signal intensity of biradicals follow Curie behavior down to 4 K ascertaining the triplet ground state or its near-degeneracy with the singlet state. UV-vis studies of 1-5 show characteristic differences in the extinctions of n-pi transitions around 600 nm. Both biradicals 1 and 2 were crystallized in monoclinic space groups C2/c and P2(1)/a with the intraradical distances 1.54 and 1.47 nm, respectively. Computational studies of the biradicals 1, 2, and 1,3-bis[4-(1-oxyl-3-oxo-4,4,5,5-tetramethylimidazolin-2-yl)phenylethynyl)]benzene (T) were performed by the AM1/CAS(8,8) method to calculate the singlet-triplet (DeltaEST) energy difference and the spin density distribution. Results show that the position of the pyridyl nitrogen in 1 and 2 in comparison with T does not alter the triplet ground-state spin multiplicities supporting the obtained experimental results.     

On the interaction of bovine serum albumin with ionic surfactants: temperature induced EPR changes of a maleimide nitroxide reflect local protein dynamics and probe solvent accessibility

The interaction of bovine serum albumin (BSA) with the ionic surfactants sodium dodecylsulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) was studied by electron paramagnetic resonance (EPR) spectroscopy of spin label covalently bound to the single free thiol group of the protein. EPR spectra simulation allows to monitor the protein dynamics at the labeling site and to estimate the changes in standard Gibbs free energy, enthalpy and entropy for transferring the nitroxide side chain from the more motionally restricted to the less restricted component. Whereas SDS and CTAC showed similar increases in the dynamics of the protein backbone for all measured concentrations, HPS presented a smaller effect at concentrations above 1.5mM. At 10mM of surfactants and 0.15 mM BSA, the standard Gibbs free energy change was consistent with protein backbone conformations more expanded and exposed to the solvent as compared to the native protein, but with a less pronounced effect for HPS. In the presence of the surfactants, the enthalpy change, related to the energy required to dissociate the nitroxide side chain from the protein, was greater, suggesting a lower water activity. The nitroxide side chain also detected a higher viscosity environment in the vicinity of the paramagnetic probe induced by the addition of the surfactants. The results suggest that the surfactant-BSA interaction, at higher surfactant concentration, is affected by the affinities of the surfactant to its own micelles and micelle-like aggregates. Complementary DLS data suggests that the temperature induced changes monitored by the nitroxide probe reflects local changes in the vicinity of the single thiol group of Cys-34 BSA residue.     

A metabolically engineered spin-labeling approach for studying glycans on cells

Metabolic glycan engineering (MGE) coupled with nitroxide spin-labeling (SL) was utilized to investigate the heterogeneous environment of cell surface glycans in select cancer and normal cells. This approach exploited the incorporation of azides into cell surface glycans followed by a click reaction with a new nitroxide spin label. Both sialic acid and N-acetylglucosamine (GlcNAc) were targeted for spin labelling. Although each of these moieties experiences a diverse and heterogeneous glycan environment, their EPR spectra and hence mobility are both characterized as a linear combination of two distinct spectra where one component reflects a highly mobile or uncrowded micro-environment with the second component reflecting more restricted motion, reflective of increased crowding and packing within the glycocalyx. What differs among the spectra of the targeted glycans is the relative percentage of each component, with sialic acid moieties experiencing on average an ∼80% less crowded environment, where conversely GlcNAc/GalNAz labeled sites reported on average a ∼50% more crowded environment. These distinct environments are consistent with the organization of sugar moieties within cellular glycans where some residues occur close to the cell membrane/protein backbone (i.e. more restricted) and others are more terminal in the glycan (i.e. more mobile). Strikingly, different cell lines displayed varied relative populations of these two components, suggesting distinctive glycan packing, organization, and composition of different cells. This work demonstrates the capability of SDSL EPR to be a broadly useful tool for studying glycans on cells, and interpretation of the results provides insights for distinguishing the differences and changes in the local organization and heterogeneity of the cellular glycocalyx.

Metabolic glycan engineering (MGE) coupled with nitroxide spin-labeling (SL) was utilized to investigate the heterogeneous environment of cell surface glycans in select cancer and normal cells.     

Determination of the relevant magnetic interactions in low-dimensional molecular materials: the fundamental role of single crystal high frequency EPR

A new one-dimensional copper(II) complex with formula [Cu(hfac)(2)(N(3)TEMPO)](n) (hfac = hexafluoroacetylacetonate and N(3)TEMPO = 4-azido-2,2,6,6-tetramethylpiperidine-1-oxyl) has been synthesized and investigated by X-ray crystallography, magnetometry and multifrequency single crystal EPR. The system crystallizes in the P1 space group with two non equivalent copper(II) ions in the unit cell, the two nitroxide radicals being coordinated to Cu(1) in axial positions. The copper(II) ions are bridged by N(3)TEMPO radicals resulting in a zig-zag chain structure. The magnetic susceptibility data were at first satisfactorily modeled assuming an alternating spin chain along the monodimensional covalent skeleton, with a ferromagnetic interaction between Cu(1) and the nitroxide moieties and a weaker antiferromagnetic interaction between these and Cu(2) (J(1) = -13.8 cm(-1), J(2) = +2.4 cm(-1)). However, single crystal EPR studies performed at the X- and W-band clearly demonstrate that the observed magnetic monodimensional character of the complex is actually due to the intermolecular contacts involving N(3)TEMPO ligands. This prompted us to fit the magnetic data using a consistent model, pointing out the fundamental role of single crystal EPR data in defining a correct model to describe the magnetic properties of molecular low dimensional systems.     

Studies toward the synthesis of 4-(2-R-ethyl)amino-2,2,5,5-tetramethyl-3-imidazoline 1-oxyls. nucleophilic substitution of bromide in the N-Alkyl chain of the 1,2,4-oxadiazol-2-one precursor

A synthetic approach to access the new nitroxides of the amidine type exhibiting pH-dependent EPR spectra through substitution of a halide in the exo-N-halogenoalkyl chain of 1-(2-bromoethyl)-6-oxyl-5,5,7,7-tetramethyltetrahydroimidazo[1,5-b][1,2,4]oxadiazol-2-one is reported. In this approach, an oxycarbonyl moiety of the oxadiazolone heterocycle plays the role of a "protecting group" for the amidine functionality. A nucleophilic cleavage of the oxadiazolone heterocycle under mild nonbasic conditions, applicable to substrates bearing substituents vulnerable to attack by strong basic nucleophiles, is elaborated. The approach allows for the new amidine nitroxides bearing various functional groups (e.g., such as CN, N3, NH2, COOEt) to be synthesized. A series of nitroxides obtained through the Staudinger/intermolecular aza-Wittig reaction of the azido derivative is also described. The nitroxides synthesized here were found to have pH-dependent two-component EPR spectra indicative of a slow, on the EPR time scale, R* left arrow over right arrow R*H+ chemical exchange, and pKa values ranging from 2.8 to 12.5 units. The guanidine derivatives synthesized in this work show the highest pKa values (pKa = 10.2 and 12.5, respectively) ever reported for the nitroxide pH-probes of a "basic type".     

Self-assembling of phenothiazine compounds investigated by small-angle X-ray scattering and electron paramagnetic resonance spectroscopy

Small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) have been carried out to investigate the structure of the self-aggregates of two phenothiazine drugs, chlorpromazine (CPZ) and trifluoperazine (TFP), in aqueous solution. In the SAXS studies, drug solutions of 20 and 60 mM, at pH 4.0 and 7.0, were investigated and the best data fittings were achieved assuming several different particle form factors with a homogeneous electron density distribution in respect to the water environment. Because of the limitation of scattering intensity in the q range above 0.15 A(-1), precise determination of the aggregate shape was not possible and all of the tested models for ellipsoids, cylinders, or parallelepipeds fitted the experimental data equally well. The SAXS data allows inferring, however, that CPZ molecules might self-assemble in a basis set of an orthorhombic cell, remaining as nanocrystallites in solution. Such nanocrystals are composed of a small number of unit cells (up to 10, in c-direction), with CPZ aggregation numbers of 60-80. EPR spectra of 5- and 16-doxyl stearic acids bound to the aggregates were analyzed through simulation, and the dynamic and magnetic parameters were obtained. The phenothiazine concentration in EPR experiments was in the range of 5-60 mM. Critical aggregation concentration of TFP is lower than that for CPZ, consistent with a higher hydrophobicity of TFP. At acidic pH 4.0 a significant residual motion of the nitroxide relative to the aggregate is observed, and the EPR spectra and corresponding parameters are similar to those reported for aqueous surfactant micelles. However, at pH 6.5 a significant motional restriction is observed, and the nitroxide rotational correlation times correlate very well with those estimated for the whole aggregated particle from SAXS data. This implies that the aggregate is densely packed at this pH and that the nitroxide is tightly bound to it producing a strongly immobilized EPR spectrum. Besides that, at pH 6.5 the differences in motional restriction observed between 5- and 16-DSA are small, which is different from that observed for aqueous surfactant micelles.