Known since the ancient times, cotton continues to be one of the essential materials for the human civilization. Cotton fibers are almost pure cellulose and contain both crystalline and amorphous nanodomains with different physicochemical properties. While understanding of interactions between the individual cellulose chains within the crystalline phase is important from a perspective of mechanical properties, studies of the amorphous phase lead to characterization of the essential transport parameters, such as solvent diffusion, dyeing, drug release, and toxin absorption, as well as more complex processes of enzymatic degradation. Here, we describe the use of spin probe electron paramagnetic resonance methods to study local polarity and heterogeneous viscosity of two types of unprocessed cotton fibers, G. hirsutum and G. barbadense, harvested in the State of North Carolina, USA. These fibers were loaded with two small molecule nitroxide probes that differ in polarity—Tempo and its more hydrophilic derivative Tempol—using a series of polar and non-polar solvents. The electron paramagnetic resonance spectra of the nitroxide-loaded cotton fibers were analyzed both semi-empirically and by least-squares simulations using a rigorous stochastic theory of electron paramagnetic resonance spectra developed by Freed and coworkers. A software package and least-squares fitting protocols were developed to carry out automatic simulations of multi-component electron paramagnetic resonance spectra in both first-derivative and the absorption forms at multiple resonance frequencies such as X-band (9.5 GHz) and W-band (94.3 GHz). The results are compared with the preceding electron paramagnetic resonance spin probe studies of a commercial bleached cotton sheeting carried out by Batchelor and coworkers. One of the results of this study is a demonstration of a co-existence of cellulose nanodomains with different physicochemical properties such as polarity and microviscosity that are affected by solvents and temperature. Spin labeling studies also revealed a macroscopic heterogeneity in the domain distribution along the cotton fibers and a critical role the cuticular layer is playing as a barrier for spin probe penetration. Finally but not lastly, the simultaneous multi-component least-squares simulation method of electron paramagnetic resonance spectra acquired at different resonant frequencies and the display forms (e.g., absorption and first-derivative displays) and the strategy of spectral parameter sharing could be potentially applicable to other heterogeneous biological systems in addition to the cotton fibers studies here.

•Lipid-embedded oligomeric proteins are not readily amenable to structural methods.•DEER and ssNMR employ the same preparation protocol of membrane protein samples.•Geometry of ASR oligomers is derived from the analysis of a multispin DEER signal.•Significant refinement of ASR ssNMR structure is achieved by adding DEER constraints.•Combined DEER + ssNMR can determine the structures of membrane protein assemblies.Oligomerization of membrane proteins is common in nature. Here, we combine spin-labeling double electron–electron resonance (DEER) and solid-state NMR (ssNMR) spectroscopy to refine the structure of an oligomeric integral membrane protein, Anabaena sensory rhodopsin (ASR), reconstituted in a lipid environment. An essential feature of such a combined approach is that it provides structural distance restraints spanning a range of ca 3–60 Å while using the same sample preparation (i.e., mutations, paramagnetic labeling, and reconstitution in lipid bilayers) for both ssNMR and DEER. Direct modeling of the multispin effects on DEER signal allowed for the determination of the oligomeric order and for obtaining long-range DEER distance restraints between the ASR trimer subunits that were used to refine the ssNMR structure of ASR. The improved structure of the ASR trimer revealed a more compact packing of helices and side chains at the intermonomer interface, compared to the structure determined using the ssNMR data alone. The extent of the refinement is significant when compared with typical helix movements observed for the active states of homologous proteins. Our combined approach of using complementary DEER and NMR measurements for the determination of oligomeric structures would be widely applicable to membrane proteins where paramagnetic tags can be introduced. Such a method could be used to study the effects of the lipid membrane composition on protein oligomerization and to observe structural changes in protein oligomers upon drug, substrate, and co-factor binding.Download high-res image (115KB)Download full-size image

Spin probe and spin labeling Electron Paramagnetic Resonance methods are indispensable research tools for solving a wide range of bioanalytical problems—from measuring microviscosity and polarity of phase-separated liquids to oxygen concentrations in tissues. One of the emerging uses of spin probes are the studies of proton transfer-related and surface electrostatic phenomena. The latter Electron Paramagnetic Resonance methods rely on molecular probes containing an additional functionality capable of reversible ionization (protonation, in particular) in the immediate proximity to an Electron Paramagnetic Resonance-active reporter group, such as (N–O•) for nitroxides. The consequent formation of protonated and nonprotonated nitroxide species with different magnetic parameters (Aiso, giso) could be readily distinguished by Electron Paramagnetic Resonance. Bioanalytical Electron Paramagnetic Resonance studies employing pH-sensitive paramagnetic probes typically involve determination of the equilibrium constant (pKa) between the protonated and nonprotonated forms of the nitroxide. However, any chemical equilibrium involving charged species, such as ionization of acids and bases, and so the reversible protonation of the nitroxide, is known to be affected by an ionic strength of the solution. Currently, only scarce data for the effect of the solution ionic strength on the experimental pKa’s of the ionizable nitroxides can be found in the literature. Here we have carried out a series of Electron Paramagnetic Resonance titration experiments for aqueous solutions of 2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl (HMI) nitroxide known for one of the largest differences in the isotropic nitrogen hyperfine coupling constant Aiso between the protonated and nonprotonated forms. Electrolyte concentration was varied over an exceptionally large range (i.e., from 0.05 to 5.0 M) to elucidate the effect of ionic strength on the ionization constant of this pH-sensitive Electron Paramagnetic Resonance probe and the data were compared to the Debye–Hückel limiting law. Effects of the ionic strength on the magnetic parameters of the ionizable nitroxides are also discussed.

Acid-base equilibria and interfacial electrostatic properties of hydrated mesoporous and nanostructured alumina powders are determining factors for the use of these materials in heterogeneous catalysis and as a sorption media for filtration and chromatographic applications including life sciences. Here spin probe electron paramagnetic resonance spectroscopy of pH-sensitive nitroxides was employed to evaluate the surface charge and interfacial acid-base equilibria at the pore surface of mesoporous powders of α-Al2O3, γ-Al2O3, Al2O3 × nH2O, and basic γ-Al2O3 and nanostructured Al2O3 in the form of pristine materials and modified with aluminum-tri-sec-butoxide, hydroxyaluminum glycerate, and several phospholipids. A new pH-sensitive nitroxide probe, 4-dimethylamino-5,5-dimethyl-2-(4-(chloromethyl)phenyl)-2-ethyl-2,5-dihydro-1H-imidazol-1-oxyl hydrochloride semihydrate (nitroxide R1), has been synthesized and characterized. It was found that conditions of preparation of alumina powders exert strikingly large effects on the apparent pKa of nitroxides measured from electron paramagnetic resonance titration curves. Specifically, while the electron paramagnetic resonance titrations curves for the nitroxide R1 in mesoporous powders prepared from basic γ-Al2O3 and Al2O3 × nH2O were shifted by ΔpKa≈ +0.6 and up to ≈ +1.2 pH units respectively, the shift for γ-Al2O3 was found to be much higher: ΔpKa = +3.5. Assuming approximately the same ∆pH = 0.5–1.0 arising from a difference in the hydrogen ion activity between the bulk solution phase and that in a confined pore volume, the samples were ranked in the following order of descending magnitude of the effective surface electrostatic potential Ψ: mesoporous γ-Al2O3 > Al2O3 × nH2O > basic γ-Al2O3 > α-Al2O3. Conditions of the Al2O3 synthesis as well as the surface modification procedures were found to have profound effects on the interfacial electrostatic properties of hydrated samples that are likely related to the nature and concentration of the active sites on the alumina surfaces.

Anodic aluminum oxide (AAO) ceramic membranes with macroscopically aligned and hexagonally packed nanopore architecture are attractive substrates for forming nanotubular lipid bilayers as well as sorption and catalytic media because of a tunable pore diameter, robust pore structure, and low fabrication cost. Here we employed continuous wave X-band (9 GHz) EPR of two pH-sensitive nitroxide radicals to assess acid–base properties AAO membranes prepared from low-cost commercial grade aluminum and compared those with commercial Anodisc membranes from Whatman, Ltd. The AAO membranes with pore diameters ≥58 ± 8 nm showed essentially the same pH inside the pores, pHint, as the bulk external solution, pHext, over the 0.1–3.0 M range of ionic strength. However, the apparent pKa of nitroxide probes inside the pores deviated from the bulk values for the nanopores of smaller diameters of ca. 29 and 18 nm. Specifically, for the latter nanopores the values of pHint were found to be 0.5–0.8 pH unit lower than the bulk pHext. An increase in acidity of the bulk solution led to a steady decrease of the negative charge on inner surface of the 38 nm nanopores and its recharge from a negative to a positive value at pH 4.7 ± 0.1, corresponding to the point of zero charge (pzc). Overall, the EPR titration method described here could assist in characterization of meso- and nanoporous membranes for catalytic and sorption applications as well as act a support medium for self-assembled biomembrane systems.

Emerging applications of nanosized iron oxides in nanotechnology introduce vast quantities of nanomaterials into the human environment, thus raising some concerns. Here we report that the surface of γ-Fe2O3 nanoparticles 20−40 nm in diameter mediates production of highly reactive hydroxyl radicals (OH•) under conditions of the biologically relevant superoxide-driven Fenton reaction. By conducting comparative spin-trapping EPR experiments, we show that the free radical production is attributed primarily to the catalytic reactions at the nanoparticles’ surface rather than being caused by the dissolved metal ions released by the nanoparticles as previously thought. Moreover, the catalytic centers on the nanoparticle surface were found to be at least 50-fold more effective in OH• radical production than the dissolved Fe3+ ions. Conventional surface modification methods such as passivating the nanoparticles’ surface with up to 935 molecules of oleate or up to 18 molecules of bovine serum albumin per iron oxide core were found to be rather ineffective in suppressing production of the hydroxyl radicals. The experimental protocols developed in this study could be used as one of the approaches for developing analytical assays for assessing the free radical generating activity of a variety of nanomaterials that is potentially related to their biotoxicity.

The synthesis and characterization of spin-labeled phospholipids (SLP)—derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PTE)—with pH-reporting nitroxides that are covalently attached to the lipid’s polar headgroup are being reported. Two lipids were synthesized by reactions of PTE with thiol-specific, pH-sensitive methanethiosulfonate spin labels methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL) and S-4-(4-(dimethylamino)-2-ethyl-5,5-dimethyl-1-oxyl-2,5-dihydro-1H-imidazol-2-yl)benzyl methanethiosulfonate (IKMTSL). The pKa values of the IMTSL−PTE lipid measured by EPR titration in aqueous buffer/isopropyl alcohol solutions of various compositions were found to be essentially the same (pKa ≈ 2.35), indicating that in mixed aqueous/organic solvents, the amphiphilic lipid molecules could be shielded from changing bulk conditions by a local shell of solvent molecules. To overcome this problem, the spin-labeled lipids were modeled by synthesizing IMTSL- and IKMTSL−2-mercaptoethanol adducts. These model compounds yielded the intrinsic pKa0’s for IMTSL−PTE and IKMTSL−PTE in aqueous buffers as 3.33 ± 0.03 and 5.98 ± 0.03, respectively. A series of EPR titrations of IMTSL−PTE in mixed water/isopropyl alcohol solution allowed for calibrating the polarity-induced pKa shifts, ΔpKapol, vs bulk solvent dielectric permittivity. These calibration data allowed for estimating the local dielectric constant, εeff, experienced by the reporter nitroxide of the IMTSL−PTE lipid incorporated into the nonionic Triton X-100 micelles as 60 ± 5 and 57 ± 5 at 23 and 48 °C, respectively. For micelles formed from an anionic surfactant sodium dodecyl sulfate (SDS) the electrostatic-induced pKa shift, ΔpKael = 2.06 ± 0.04 units of pH, was obtained by subtracting the polarity-induced contribution. This shift yields Ψ = −121 mV electric potential of the SDS micelle surface.

This communication reports on post-processing of continuous wave EPR spectra by a digital convolution with filter functions that are subjected to differentiation or the Kramers–Krönig transform analytically. In case of differentiation, such a procedure improves spectral resolution in the higher harmonics enhancing the relative amplitude of sharp spectral features over the broad lines. At the same time high-frequency noise is suppressed through filtering. These features are illustrated on an example of a Lorentzian filter function that has a principal advantage of adding a known magnitude of homogeneous broadening to the spectral shapes. Such spectral distortion could be easily and accurately accounted for in the consequent least-squares data modeling. Application examples include calculation of higher harmonics from pure absorption echo-detected EPR spectra and resolving small hyperfine coupling that are unnoticeable in conventional first derivative EPR spectra. Another example involves speedy and automatic separation of fast and broad slow-motion components from spin-label EPR spectra without explicit simulation of the slow motion spectrum. The method is illustrated on examples of X-band EPR spectra of partially aggregated membrane peptides.

A first thiol-specific pH-sensitive nitroxide spin-label of the imidazolidine series, methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL), has been synthesized and characterized. X-Band (9 GHz) and W-band (94 GHz) EPR spectral parameters of the new spin-label in its free form and covalently attached to an amino acid cysteine and a tripeptide glutathione were studied as a function of pH and solvent polarity. The pKa value of the protonatable tertiary amino group of the spin-label was found to be unaffected by other ionizable groups present in side chains of unstructured small peptides. The W-band EPR spectra were shown to allow for pKa determination from precise g-factor measurements. Is has been demonstrated that the high accuracy of pKa determination for pH-sensitive nitroxides could be achieved regardless of the frequency of measurements or the regime of spin exchange: fast at X-band and slow at W-band. IMTSL was found to react specifically with a model protein, iso-1-cytochrome c from the yeast Saccharomyces cerevisiae, giving EPR spectra very similar to those of the most commonly employed cysteine-specific label MTSL. CD data indicated no perturbations to the overall protein structure upon IMTSL labeling. It was found that for IMTSL, giso correlates linearly with Aiso, but the slopes are different for the neutral and charged forms of the nitroxide. This finding was attributed to the solvent effects on the spin density at the oxygen atom of the NO group and on the excitation energy of the oxygen lone-pair orbital.

This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure–function studies of membrane proteins by solid-state NMR.

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes.

Anodic aluminum oxide substrates with macroscopically aligned homogeneous nanopores of 80 nm in diameter enable two-dimensional, solid-state nuclear magnetic resonance studies of lipid-induced conformational changes of uniformly 15N-labeled Pf1 coat protein in native-like bilayers. The Pf1 helix tilt angles in bilayers composed of two different lipids are not entirely governed by the membrane thickness but could be rationalized by hydrophobic interactions of lysines at the bilayer interface. The anodic aluminum oxide alignment method is applicable to a broader repertoire of lipids versus bicelle bilayer mimetics currently employed in solid-state nuclear magnetic resonance of oriented samples, thus allowing for elucidation of the role played by lipids in shaping membrane proteins.