Electron Spin Echo Envelope Modulation Technique Research Articles

Radiation-Induced Stable Radicals in Calcium Phosphates: Results of Multifrequency EPR, EDNMR, ESEEM, and ENDOR Studies

This article presents the results of a study of radiation-induced defects in various synthetic calcium phosphate (CP) powder materials (hydroxyapatite—HA and octacalcium phosphate—OCP) by electron paramagnetic resonance (EPR) spectroscopy at the X, Q, and W-bands (9, 34, 95 GHz for the microwave frequencies, respectively). Currently, CP materials are widely used in orthopedics and dentistry owing to their high biocompatibility and physico-chemical similarity with human hard tissue. It is shown that in addition to the classical EPR techniques, other experimental approaches such as ELDOR-detected NMR (EDNMR), electron spin echo envelope modulation (ESEEM), and electron-nuclear double resonance (ENDOR) can be used to analyze the electron–nuclear interactions of CP powders. We demonstrated that the value and angular dependence of the quadrupole interaction for 14N nuclei of a nitrate radical can be determined by the EDNMR method at room temperature. The ESEEM technique has allowed for a rapid analysis of the nuclear environment and estimation of the structural positions of radiation-induced centers in various crystal matrices. ENDOR spectra can provide information about the distribution of the nitrate radicals in the OCP structure.

Electron spin echo envelope modulation of molecular motions of deuterium nuclei

Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy is a powerful technique for the study of hyperfine interactions between an unpaired electron and nearby nuclei in solids, and is employed in quantitative structural studies. Here, we describe the use of ESEEM to study the slow motion of deuterium nuclei using their nuclear quadrupole resonance (NQR) line shapes. Two ESEEM techniques were employed: the conventional three-pulse ESEEM experiment, π/2 – τ – π/2 – T– π/2 – τ – echo, and the four-pulse ESEEM, π/2 – τ – π/2 – T/2 – π – T/2 – π/2 – τ – echo, with the time variable T scanned in both cases. The nitroxide free radical 4-tert-butyliminomethyl-2,2,5,5-tetramethyl(d12)-3-imidazoline-1-oxyl with four deuterated methyl groups was investigated in a glassy ortho-terphenyl matrix over a wide temperature range. It was shown that four-pulse ESEEM allowed measurement of the nearly pure 2H NQR line shape. Between 90K and 120K, the ESEEM spectra change drastically. At low temperatures, four-pulse ESEEM spectra show a Pake-like pattern, which evolves into a single line at higher temperatures, which is typical for NQR of rotating methyl CD3 groups. Comparison with literature data on NQR allows estimation of the reorientation rate, k. At ∼100K, where the spectral changes are most pronounced, k was found to be ∼105s−1. The spectral linewidths for the three-pulse ESEEM were found to decrease similarly with increasing temperature; so the three-pulse technique is also capable to detect motion of this type. The ESEEM approach, along with site-directed spin labeling, may be useful for detection of motional transitions near the spin labels in biological systems, when information on motion is required in a wide temperature range.

Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer.

Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane-bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a (2) H-labeled d8 -Val overexpressed membrane protein in a membrane mimetic environment. The membrane-bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different α-helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated α-helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be α-helical in a more native-like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X-ray crystallography and/or NMR spectroscopy.

Probing the Secondary Structure of Membrane Protein using Bacterial Expression System and Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

Membrane proteins possess a variety of functions essential to the survival of organisms. However, due to the inherent hydrophobic property, it is extremely difficult to gain the structural information. Previously, we probed the structure of an α-helical component of a small model membrane peptide, AchR M2δ, by detecting 2H modulation from a 2H-labeled Val side chain that was 3 or 4 residues away from a nitroxide spin label using ESEEM spectroscopy. In this study, for the first time, ESEEM technique was applied to determine the local secondary structure of a biological membrane protein system using bacterial expression method for incorporating the 2H-labeled Val side chain both in micelle and lipid bilayer environment. An integral membrane protein, KCNE1 with known 3D structure, was utilized as a model membrane protein to test the feasibility and validity of this ESSEM technique. The side chain of valine at position 21 (outside domain), 50 (transmembrane domain), and 95 (cytoplasmic domain) was 2H labeled (denoted as ith position). An MTSL nitroxide spin label was positioned at 2, 3, and 4 residues away (denoted i+2 to i+4, respectively) from 2H-labeled Val residue of each domain. Our results indicated that i+3 and i+4 positions have a significant 2H modulation while 2H modulation is absent at i+2 position, which is consistent with the 3.6 residue/turn periodicity of the α-helix. These results are also consistent with the micelle structure of KCNE1. This ESEEM technique was further validated using the protein incorporated into lipid bilayer of DMPC/DPC bicelle and the transmembrane domain was probed to be α-helical as well. This ESEEM technique is very powerful, efficient, and quick to examine the local secondary structure of any protein system without size limitation.

Probing the Secondary Structure of Membrane Proteins with the Pulsed EPR ESEEM Technique

A novel approach is being developed to probe the secondary structure of membrane proteins and peptides qualitatively utilizing the three-pulse Electron Spin Echo Envelope Modulation (ESEEM) pulse sequence. In order to demonstrate the practicality of this biophysical technique, the M2delta subunit of AChR (α-helical), KAGAKI (β-sheet) and VRL8 (310-helix) peptides were incorporated into phospholipids bicelles to probe their secondary structure with ESSEM spectroscopy. Utilizing site-directed spin-labeling (SDSL) coupled with deuterated amino acid labeling of the peptides, the corresponding ESEEM spectra reveal characteristic patterns for α-helix, β-sheet and 310-helical structures. This ESSEM secondary structural approach can be used with different deuterated amino acids and provide pertinent qualitative structural information on membrane proteins in a short period of time (10 minutes) with small amounts of sample (30 μg).

Topology of the Trans-Membrane Peptide WALP23 in Model Membranes under Negative Mismatch Conditions

The organization and orientation of membrane-inserted helices is important for better understanding the mode of action of membrane-active peptides and of protein-membrane interactions. Here we report on the application of ESEEM (electron spin-echo envelope modulation) and DEER (double electron-electron resonance) techniques to probe the orientation and oligomeric state of an α-helical trans-membrane model peptide, WALP23, under conditions of negative mismatch between the hydrophobic cores of the model membrane and the peptide. Using ESEEM, we measured weak dipolar interactions between spin-labeled WALP23 and (2)H nuclei of either the solvent (D2O) or of lipids specifically deuterated at the choline group. The ESEEM data obtained from the deuterated lipids were fitted using a model that provided the spin label average distance from a layer of (2)H nuclei in the hydrophilic region of the membrane and the density of the (2)H nuclei in the layer. DEER was used to probe oligomerization through the dipolar interaction between two spin-labels on different peptides. We observed that the center of WALP23 does not coincide with the bilayer midplane and its N-terminus is more buried than the C-terminus. In addition, the ESEEM data fitting yielded a (2)H layer density that was much lower than expected. The DEER experiments revealed the presence of oligomers, the presence of which was attributable to the negative mismatch and the electrostatic dipole of the peptide. A discussion of a possible arrangement of the individual helices in the oligomers that is consistent with the ESEEM and DEER data is presented.

Probing the Structure of Membrane Proteins with ESEEM and DEER Pulsed EPR Techniques

Limited structural and dynamic information on membrane proteins and peptides exist. New biophysical/structural biology methods are needed to probe these systems in a lipid bilayer. The Lorigan lab is applying unique hybrid NMR and spin-label EPR spectroscopic techniques to study membrane proteins. Magnetic resonance spectroscopic data of 15N-, 2H-labeled and/or spin-labeled membrane proteins incorporated into vesicles and bicelles will be presented. State-of-the-art pulsed EPR techniques such as Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy, and Double Electron-Electron Resonance (DEER) spectroscopy will be used. The ESEEM technique can determine short to medium range distances (out to about 8 A) between a site-specific nitroxide spin label and a nearby NMR-active isotopic labeled residue for a variety of different peptides and proteins which ultimately can be used to determine the difference between an α-helical and β-sheet secondary structure. DEER can be used to measure distances between 2 spin labels out to about 70 A. We have shown a huge improvement is sensitivity with DEER measurements at Q-band when compared to X-band.

Investigating the Structural and Dynamic Properties of Membrane Proteins with Solid-State NMR, CW-EPR, ESEEM, and DEER Spectroscopic Techniques

Currently, we have very limited structural and dynamic information on membrane proteins and peptides. New spectroscopic methods are needed to probe these systems in a lipid bilayer. In order to address these issues, the Lorigan lab developing unique hybrid solid-state NMR and spin-label EPR spectroscopic techniques. Magnetic resonance spectroscopic data of 15N-, g2H-labeled and/or spin-labeled membrane proteins incorporated into vesicles and bicelles will be presented. State-of-the-art solid-state NMR and pulsed EPR techniques such as Magic Angle Spinning (MAS) NMR, Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy, and Double Electron-Electron Resonance (DEER) spectroscopy will be used. The ESEEM technique can determine short to medium range distances (out to about 8 Å) between a site-specific nitroxide spin label (MTSL) and a nearby NMR-active isotopic labeled residue for a variety of different peptides and proteins which ultimately can be used to determine the difference between an α-helical and β-sheet secondary structure. DEER can be used to measure distances between 2 spin labels out to about 70 Å. The advantages and disadvantages of applying solid-state NMR and EPR spectroscopy to probe the structural and dynamic properties of membrane proteins will be discussed.

Probing the Structure of Membrane Proteins Using Pulsed EPR ESEEM Spectroscopy

Current methods used for determining the secondary structure of membrane proteins require large sample sizes and have long data acquisition times. Therefore using pulsed EPR spectroscopic technique of Electron Spin Echo Envelope Modulation (ESEEM) is advantageous due to the requirement of less sample, short data acquisition, and high sensitivity. Using this technique, we can determine short to medium range distances (up to 8 A) between a site-specific nitroxide spin label (MTSL) and a nearby NMR-active isotopic labeled residue for a variety of peptides and proteins which ultimately determine the difference between an α-helical and β-sheet secondary structure. The information can be obtained using a three-pulse ESEEM sequence which allows for the detection of the fundamental nuclear spin transitions. It is possible to calculate the radial distance between a paramagnetic electron and a weakly coupled nucleus because the modulation depth produced by a weakly dipolar-coupled nucleus is inversely proportional to the radial distance. This new method is applied to two different membrane peptides, M2δ AChR, and KIGAKI, which have α-helical and β-sheet structural components respectively. A nitroxide spin probe is attached to a specific Cys residue at a given position I, and a 2H isotopic labeled residue is attached to a nearby residue (i + 3). The corresponding data shows that an α-helical structure will yield a very large 2H peak, whereas a β-sheet will yield a much smaller 2H peak, thus a difference can be observed between the two secondary structures. The ESEEM technique requires less protein sample (about 75 μg) and data acquisition only takes about 1 hour which makes it a simple, powerful, and effective technique in the determination of the secondary structure of a membrane protein.

Comparison of CW-EPR and ESEEM technique for determination of water permeability profile in liposome membranes

The hydrophobicity profile across a membrane is an important parameter of biological membranes as it defines the barrier for permeation of polar molecules into the cell interior and of the stability of transmembrane proteins. In this work, the hydrophobicity profile was measured by conventional continuos-wave electron paramagnetic resonance (CW-EPR) and by electron spin echo envelope modulation (ESEEM) in two liposome suspensions with different ionic strengths in order to compare the sensitivity of both methods for the determination of the water penetration depth into the liposome membrane. Multilamellar liposomes from egg-phosphatidylcholine, cholesterol and dicetyl phosphate were prepared in deuterium oxide, buffered with phosphate buffer with or without NaCl at the phosphate concentrations 0.1 or 0.01 M. Liposomes were spin labeled with stearic acids spin probes with the nitroxide group at different positions on the acyl chain. The deuterium penetration depth was measured by the ESEEM technique and the signal was analyzed in the frequency domain (Fourier transform [FT]-ESEEM). The ratio of the intensities of FT-ESEEM lines at deuterium and hydrogen Larmor frequencies was used as a measures of the water penetration depth. It was also indirectly determined from the hyperfine splitting constants in frozen and in fluid state, measured by CW-EPR, as they depend on the polarity of the nitroxide group environment. The comparison of the two techniques demonstrated that FT-ESEEM is more sensitive to the changes on the surface of the membrane and thus more appropriate for the determination of the hydrophobic barrier than the conventional CW-EPR method.

ESE-ENDOR and ESEEM Characterization of Water and Methanol Ligation to a Dinuclear Mn(III)Mn(IV) Complex

Interactions of water and methanol with a mixed valence Mn(III)Mn(IV) complex are explored with 1H electron spin echo (ESE)-electron nuclear double resonance (ENDOR) and 1H and 2H ESE envelope modulation (ESEEM). Derivatives of the (2-OH-3,5-Cl2-SALPN)2 Mn(III)Mn(IV) complex are ideal for structural and spectroscopic modeling of water binding to multinuclear Mn complexes in metalloproteins, specifically photosystem II (PSII) and manganese catalase (MnCat). Using ESE-ENDOR and ESEEM techniques, 1H hyperfine parameters are determined for both water and methanol ligated to the Mn(III) ion of the complex. The protons of water directly bound to Mn(III) are inequivalent and exhibit roughly axial dipolar hyperfine interactions (Tdip = 8.4 MHz and Tdip = 7.4 MHz), permitting orientations and radial distances to be determined using a model where the proton experiences a point dipole interaction with each Mn ion. General equations are given for the components of the rhombic dipolar hyperfine interaction between a proton and a spin coupled dinuclear metal cluster. The observed ENDOR pattern is from water protons 2.65 and 2.74 Å from the Mn(III) which make an Mn(IV)−Mn(III)−H angle of ∼160°. For the alcohol proton in the analogous methanol bound complex, a 2.65 Å Mn(III)−H distance is observed. Three pulse 2H ESEEM gives best fit Mn(III)−2H(1H) radial distances of 3.0, 3.5, and 4.0 Å for the three methyl deuterons in this complex.

Determination of hyperfine interaction matrix principal values and principal axis orientations in an orientationally disordered solid: A multifrequency electron spin echo envelope modulation study of nitrogen-15 in a copper(II)–15N–imidazole complex

A comprehensive experimental study of the magnetic field dependences of ESEEM (electron spin echo envelope modulation) for a spin one-half nucleus in an orientationally disordered solid is presented. Modulation effects from the remote (unligated) nitrogen of 15N-labeled imidazole in a Cu(II)–diethylenetriamine–15N-imidazole complex were registered at electron spin excitation frequencies in the 4.3 to 11.5 GHz range, which encompasses the ‘‘match range’’ of the Cu(II)–15N system under study. Field dependent trends in the ESEEM spectra—trends in spectral amplitudes, linewidths, and frequencies—are analyzed to obtain the magnitude and relative signs of the isotropic and axially symmetric hyperfine coupling constants (and to place an upper bound on the rhombic coupling). The relative utility of each of these trends for measuring hyperfine interactions in orientationally disordered solids is discussed. The orientation of the unique axis of the Cu(II)–15N hyperfine interaction matrix within the Cu(II) electron spin magnetic axis system is obtained from the field dependence of the ESEEM frequencies observed with orientationally selective excitation. The results of this study are compared to those previously obtained for relevant Cu(II)–15N systems by a variety of monofrequency ESEEM techniques.

Orientation-selective 14N electron spin echo envelope modulation (ESEEM): The determination of 14N quadrupole coupling tensor principal axis orientations in orientationally disordered solids

We present an orientation-selective ESEEM (electron spin echo envelope modulation) technique. From a measurement of the variation of the amplitudes of modulation components at 14N pure quadrupole frequencies as a function of irradiation position within an EPR (electron paramagnetic resonance) powder pattern, we determine the orientation of the principal axes of the quadrupole coupling tensor relative to the principal axes of the tensor which governs the dispersion of the EPR spectrum in an orientationally disordered sample. The pure quadrupole frequencies appear when the EPR frequency is selected such that the nuclear Zeeman and the hyperfine interaction are approximately equal in magnitude. We have applied the method to a mercaptoethanol complex of myoglobin in which the pure quadrupole frequencies originate from 14N in the proximal imidazole ring. Our results enable us to validate the assignment of the quadrupole modulations to the metal-coordinated nitrogen of the imidazole ring, to correlate the principal axes with the principal values of the quadrupole coupling tensor, and to determine the orientation of the imidazole ring relative to the principal axes of the (low-spin) Fe(III) g matrix. These findings are discussed and compared with results of previous studies.

An electron spin-echo envelope modulation study of 14N nuclear hyperfine and quadrupole coupling in copper(II)/nickel(II) bis N,N-di- n-butyl-dithiocarbamate)

The electron spin-echo envelope modulation technique (ESEEM) is used to determine the 14N hyperfine and quadrupole tensors in copper-doped nickel(II) bis( N,N-di- n-butyl-dithiocarbamate). The line positions and peak intensities of the Fourier-transformed ESEEM spectra are interpreted using simulated spectra, obtained by diagonalizing the full spin-Hamiltonian matrix. The 14N quadrupole tensor can be accounted for theoretically by extended Hückel molecular orbital calculations. For the interpretation of the 14N hyperfine tensor, however, an unrestricted molecular orbital calculation is necessary. Therefore, one should be cautions in drawing conclusions about spin-density distributions from small hyperfine couplings of nuclei in higher coordination spheres.