Orientation of TOAC amino-acid spin labels in alpha-helices and beta-strands

The orientation of alpha-helices or beta-strands, e.g., in membranes, can be determined from EPR order parameters of (2,2,6,6-tetramethyl-piperidine-1-oxy-4-amino-4-carboxylic acid) TOAC amino-acid spin labels incorporated in the polypeptide backbone. This requires knowledge of the inclination of the nitroxide axes, relative to the alpha-helix or beta-strand axis. Crystal structures of TOAC-containing peptides are used to derive the spin-label orientation relative to refined alpha-poly-l-alanine and beta-poly-l-alanine structures. The spin-label z-axes of the two mirror-image TOAC twist-boat conformers are inclined at 13+/-2 degrees and 65+/-3 degrees , respectively, to the alpha-helix axis, or at 25+/-3 degrees and 32+/-3 degrees to the beta-strand axis.     

Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes

Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis–Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.     

Spin-Label EPR for Determining Polarity and Proticity in Biomolecular Assemblies: Transmembrane Profiles

Hyperfine couplings and g-values of nitroxyl spin labels are sensitive to polarity and hydrogen bonding in the environment probed. The dependences of these electronic paramagnetic resonance (EPR) properties on environmental dielectric permittivity and proticity are reviewed. Calibrations are given, in terms of the Block–Walker reaction field and local proton donor concentration, for the nitroxides that are commonly used in spin labeling of lipids and proteins. Applications to studies of the transverse polarity profiles in lipid bilayers, which constitute the permeability barrier of biological membranes, are reviewed. Emphasis is given to parallels with the permeation profiles of oxygen and nitric oxide that are determined from spin-label relaxation enhancements by using nonlinear continuous-wave EPR and saturation recovery EPR, and with permeation profiles of D₂O that are determined by using ²H electron spin echo envelope modulation spectroscopy.     

Multifrequency simulations of the EPR spectra of lipid spin labels in membranes

Simulations are performed of 34- and 9-GHz EPR spectra, together with 94-GHz EPR spectra, from phospholipid probes spin-labelled at the C4-C14 positions of the sn-2 chain, in liquid-ordered and gel-phase membranes of dimyristoyl phosphatidylcholine with high and low cholesterol contents. The multifrequency simulation strategy involves: (i) obtaining partially averaged spin-Hamiltonian tensors from fast-motional simulations of the 94-GHz spectra; (ii) performing slow-motional simulations of the 34- and 9-GHz spectra by using these pre-averaged tensors with the stochastic Liouville formalism; (iii) constructing, by simulation, slow-motional calibrations for the differences, DeltaA(zz)(qx) and Deltag(zz)(qx), in effective A(zz)-hyperfine splittings and g(zz)-values between 34- (or 94-GHz) and 9-GHz spectra; (iv) using such calibrations for DeltaA(zz)(qx) and Deltag(zz)(qx) and dynamic parameters from stage (ii) as a guide to adjust the extent of pre-averaging of the spin-Hamiltonian tensors; and (v) repeating the 34- and 9-GHz simulations of stage (ii). By using this scheme it is possible to obtain consistent values of the rotational diffusion coefficients, D(R perpendicular) and D(R//), and the long-axis order parameter, S(zz), that characterize the slow axial motion of the lipid chains, from spectra at both 34 and 9GHz. Inclusion of spectra at 34GHz greatly improves precision in determining the D(R//) element of the slow diffusion tensor in these systems.     

Molecular dynamics and spatial distribution of TOAC spin-labelled peptaibols studied in glassy liquid by echo-detected EPR spectroscopy

2,2,6,6-tetramethylpiperidine-l-oxyl-4-carboxylic acid (TOAC) spin-labelled analogues of the Aib-rich peptide (peptaibol) Trichogin GA IV are investigated in a glassy methanol/glycerol (70/30 v/v%) system, using conventional CW EPR and electron spin echo spectroscopy. Echo-detected (ED) EPR spectra indicate that the labels undergo restricted orientational motion (libration). Comparison with the small molecular spin probe Tempone shows that the dynamics of peptide molecules is determined by their structure and not by the surrounding medium. At the terminal positions (position 1 and 8) TOAC residues were found to be more flexible than at the central 4th position. Instantaneous diffusion mechanism in electron spin echo, which also contributes to the ED EPR spectra, was employed for deriving the local concentration of peptaibols. This approach may serve as a tool for investigation of peptide aggregation. In the studied model glassy solution the peptaibols were found to be randomly distributed.     

Theoretical spin density in nitroxides

The evolution of the hyperfine tensors in the nitroxide series with increasing alkyl substitution on the NO group has been studied theoretically for radicals from H₂NO to C₅H₁₀NO. A projection technique has been applied to the UHF wave-functions in order to correct spin densities for quartet contamination. The magnitude of the isotropic and anisotropic couplings reflects the substitution effect already observed on the spin distribution maps, that is a spin transfer from oxygen to nitrogen when alkyl groups are substituted to hydrogens in H₂NO. The alternation of the signs of the couplings along the chain as well as the cos² γ law for the coupling constants of atoms (C or H) in β position are verified in the series. The orientation of the anisotropic tensors with respect to the chemical bonds depends on the position of the atoms in the molecule (radical site, α, β positions); it is not affected by further substitution, except for strongly asymmetric configurations.     

Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels

A highly sensitive solid state (19)F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF(3)-Phg) at different positions throughout the alpha-helical peptide chain. A simple 1-pulse (19)F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF(3)-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF(3)-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF(3)-labeled peptide analogues (with L-CF(3)-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a D-CF(3)-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF(3)-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF(3)-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of experimental conditions and dynamic regimes of the peptide.     

Resolving domains of interdigitated phospholipid membranes with 95 GHz spin labeling EPR

High-field W-band (95 GHz) electron paramagnetic resonance (EPR) study of partitioning of a small nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) in multilamellar liposomes composed from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) is described. The high-resolution spectra with a high signal-to-noise ratio were combined with automated least-squares simulation analysis to derive accurate partitioning coefficients of TEMPO in the membrane lipid phase and to follow the membrane phase transitions. The isotropic magnetic parameters, g_iso and A_iso were used to characterize the average polarity the spin label is experiencing in the membrane. We also report an empirical correlation between g_iso and A_iso for a set of protic and aprotic solvents and use this correlation to assign domains formed by interdigitation of DPPC bilayer under a high ethanol concentration of 1.2 M.     

Anisotropic motion effects in CW non-linear EPR spectra: relaxation enhancement of lipid spin labels

Continuous-wave (CW) EPR measurements of enhancements in spin-lattice (T(1)-) relaxation rate find wide application for determining spin-label locations in biological systems. Often, especially in membranes, the spin-label rotational motion is anisotropic and subject to an orientational potential. We investigate here the effects of anisotropic diffusion and ordering on non-linear CW-EPR methods for determining T(1) of nitroxyl spin labels. Spectral simulations are performed for progressive saturation of the conventional in-phase, first-harmonic EPR signal, and for the first-harmonic absorption EPR signals detected 90 degrees -out-of-phase with respect to the Zeeman field modulation. Motional models used are either rapid rotational diffusion, or strong-jump diffusion of unrestricted frequency, within a cone of fixed maximum amplitude. Calculations of the T(1)-sensitive parameters are made for both classes of CW-experiment by using motional parameters (i.e., order parameters and correlation times), intrinsic homogeneous and inhomogeneous linewidth parameters, and spin-Hamiltonian hyperfine- and g-tensors, that are established from simulation of the linear CW-EPR spectra. Experimental examples are given for spin-labelled lipids in membranes.     

Probing membrane topology by high-resolution 1H-13C heteronuclear dipolar solid-state NMR spectroscopy

Membrane topology changes introduced by the association of biologically pertinent molecules with membranes were analyzed utilizing the (1)H-(13)C heteronuclear dipolar solid-state NMR spectroscopy technique (SAMMY) on magnetically aligned phospholipid bilayers (bicelles). The phospholipids (1)H-(13)C dipolar coupling profiles lipid motions at the headgroup, glycerol backbone, and the acyl chain region. The transmembrane segment of phospholamban, the antimicrobial peptide (KIGAKI)(3) and cholesterol were incorporated into the bicelles, respectively. The lipids (1)H-(13)C dipolar coupling profiles exhibit different shifts in the dipolar coupling contour positions upon the addition of these molecules, demonstrating a variety of interaction mechanisms exist between the biological molecules and the membranes. The membrane topology changes revealed by the SAMMY pulse sequence provide a complete screening method for analyzing how these biologically active molecules interact with the membrane.     

Polarity contributions to hyperfine splittings of hydrogen-bonded nitroxides--the microenvironment of spin labels

A self-consistent treatment of reaction field effects on isotropic (14)N hyperfine coupling constants of nitroxide spin labels in mixtures of polar and apolar solvents is given based on the Onsager approach. It is shown that this works reasonably well for mixtures of water or methanol with dioxane, far better than do conventional approaches using the Clausius-Mossotti relation. Association constants, K(A,h), for hydrogen bonding of protic solvents to nitroxides are derived in this way from published EPR data. A value of K(A,h) approximately 1.0 M x (-1) is argued to be reasonable for water in a hydrophobic environment. Data from spin-labelled lipids can then be used to estimate effective water concentrations in biological membranes.     

Capture and manipulation of magnetically aligned Pf1 with an aqueous polymer gel

The magnetic alignment of the Pseudomonas bacteriophage Pf1 is captured indefinitely in a gel of the aqueous triblock copolymer Pluronic F-127. In addition to preserving high-resolution liquids NMR spectra for dissolved solutes, the gel prevents the reorientation of the phage allowing mechanical manipulation of the angle between the axis of the phage alignment and the static magnetic field. The residual 2H quadrupolar couplings for several solutes dissolved in this material as a function of the angle Theta between the non-spinning sample tube and the static magnetic field are consistent with the value of P(2)(cosTheta)=(3cos(2)Theta-1)/2. The variable-angle correlation spectrum for these solutes is shown to separate residual quadrupolar effects from isotropic chemical shifts. Finally, the compatibility of Pluronic F-127 with NMR studies of aqueous biological macromolecules is demonstrated in a measurement of residual dipolar couplings in an 15N-labeled nucleic acid.     

Rotation of magnetization in unison and langevin equations for a large spin

A single-domain ferromagnetic particle is represented as a large spin (model of rotation in unison) whose stochastic dynamics is derived from a spin-boson Hamiltonian. It is shown in the Markovian limit that thermal equilibrium exists provided that the fluctuation-dissipation theorem is supplemented by a symmetry constraint which for bilinear anisotropic and nonlinear (magnetoelastic) spin-bath coupling can only be satisfied in the underdamped limit. Only for bilinear isotropic coupling (Gilbert's theory) is it satisfied identically for arbitrary damping strength. Uniaxial and cubic symmetries are considered. For a model uniaxial crystal the thermal decay rate of M and the thermal enhancement of the macroscopic quantum tunneling rate are calculated for Gilbert and magnetoelastic dissipative couplings and compared. The effects of memory are discussed.     

Nuclear spin relaxation rates in two-Leg spin ladders

Using the transfer-matrix density-matrix renormalization group method, we study the nuclear spin relaxation rate 1/T(1) in the two-leg s = 1 / 2 ladder as a function of the interchain (J( perpendicular)) and intrachain (J( parallel)) couplings. In particular, we separate the q(y) = 0 and pi contributions and show that the latter contribute significantly to the copper relaxation rate (63)(1/T(1)) in the experimentally relevant coupling and temperature range. We compare our results to both theoretical predictions and experimental measures on ladder materials.     

Simultaneous extraction of multiple orientational constraints of membrane proteins by 13C-detected N-H dipolar couplings under magic angle spinning

A 13C-detected N-H dipolar coupling technique is introduced for uniaxially mobile membrane proteins for orientation determination using unoriented samples. For proteins undergoing rigid-body uniaxial rotation in the lipid bilayer, the intrinsic equality between the dipolar coupling constants measured in unoriented samples and the anisotropic coupling measured in static oriented samples has been shown recently. Here, we demonstrate that the orientation-sensitive backbone N-H dipolar couplings can be measured with 13C detection using 2D and 3D MAS correlation experiments, so that maximal site resolution can be achieved and multiple orientational constraints can be extracted from each experiment. We demonstrate this technique on the M2 transmembrane peptide of the influenza A virus, where the N-H dipolar couplings of various residues fit to a dipolar wave for a helical tilt angle of 37 degrees , in excellent agreement with data obtained from singly 15N-labeled samples.     

Saturation recovery EPR and ELDOR at W-band for spin labels

A reference arm W-band (94 GHz) microwave bridge with two sample-irradiation arms for saturation recovery (SR) EPR and ELDOR experiments is described. Frequencies in each arm are derived from 2 GHz synthesizers that have a common time-base and are translated to 94 GHz in steps of 33 and 59 GHz. Intended applications are to nitroxide radical spin labels and spin probes in the liquid phase. An enabling technology is the use of a W-band loop-gap resonator (LGR) [J.W. Sidabras, R.R. Mett, W. Froncisz, T.G. Camenisch, J.R. Anderson, J.S. Hyde, Multipurpose EPR loop-gap resonator and cylindrical TE₀₁₁ cavity for aqueous samples at 94 GHz, Rev. Sci. Instrum. 78 (2007) 034701]. The high efficiency parameter (8.2 GW^−1/2 with sample) permits the saturating pump pulse level to be just 5 mW or less. Applications of SR EPR and ELDOR to the hydrophilic spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPONE) are described in detail. In the SR ELDOR experiment, nitrogen nuclear relaxation as well as Heisenberg exchange transfer saturation from pumped to observed hyperfine transitions. SR ELDOR was found to be an essential method for measurements of saturation transfer rates for small molecules such as TEMPONE. Free induction decay (FID) signals for small nitroxides at W-band are also reported. Results are compared with multifrequency measurements of T_1e previously reported for these molecules in the range of 2–35 GHz [J.S. Hyde, J.-J. Yin, W.K. Subczynski, T.G. Camenisch, J.J. Ratke, W. Froncisz, Spin label EPR T₁ values using saturation recovery from 2 to 35 GHz. J. Phys. Chem. B 108 (2004) 9524–9529]. The values of T_1e decrease at 94 GHz relative to values at 35 GHz.     

Polarization transfer from para-hydrogen to heteronuclei: effect of H/D substitution. The case of AA'X and A2A2'X spin systems

Upon hydrogenation from para-hydrogen (p-H(2)), hyperpolarization transfer toward a heteronucleus may be possible even if the two protons are chemically equivalent in the final product (but not magnetically equivalent), provided that J couplings with the heteronucleus exist. It is however shown (theoretically and experimentally) that this transfer effectively occurs if the spin system in the hydrogenated molecule is of the type AA'X (A and A' denoting the two protons originating from p-H(2) and X the heteronucleus) but does not occur for a spin system of the A(2)A(2)(')X type. A theory has been worked out for assessing the details of the X spectrum (multiplet patterns) in the case of ALTADENA and PASADENA experiments. Experimental verifications are provided.     

Scalings of domain wall energies in two dimensional Ising spin glasses

We study domain wall energies of two dimensional spin glasses. The scaling of these energies depends on the model's distribution of quenched random couplings, falling into three different classes. The first class is associated with the exponent theta approximately -0.28; the other two classes have theta=0, as can be justified theoretically. In contrast to previous claims, we find that theta=0 does not indicate d=d(c)(l) but rather d< or =d(c)(l), where d(c)(l) is the lower critical dimension.     

Acquisition of pure-phase diffusion spectra using oscillating-gradient spin echo

Oscillating-gradient spin echo (OGSE) diffusion experiments have long been used to measure the short-time apparent diffusion coefficient, D(app)(t), in the presence of restricted diffusion, as well as the spectrum of the slow-motion velocity autocorrelation function. In this work, we focus on two previously unexplored aspects of OGSE experiments: convection compensation and acquisition of pure-phase diffusion spectra in the presence of homonuclear scalar couplings. We demonstrate that convection compensation afforded by single-echo OGSE compares well with that in double-echo convection-compensated PGSE experiments. We also show that, in the presence of homonuclear scalar couplings, setting the OGSE echo time to 1/2J enables acquisition of pure-phase diffusion spectra and yields more reliable D estimates than mixed-phase PGSE or OGSE spectra. Pure-phase OGSE acquisition is also compatible with measurements of the apparent diffusion coefficient at an arbitrary diffusion time. These features of OGSE can be valuable in diffusion measurements of scalar-coupled small-molecule probes in cellular and other heterogeneous systems.