Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban

Electron paramagnetic resonance (EPR) was used to optimize the solid-phase peptide synthesis of a membrane-bound peptide labeled with TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid). The incorporation of this paramagnetic amino acid results in a nitroxide spin label coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by EPR. We applied this approach to phospholamban, which regulates cardiac calcium transport. The synthesis of this amphipathic 52-amino-acid membrane peptide including TOAC is a challenge, especially in the addition of TOAC and the next several amino acids. Therefore, EPR of synthetic intermediates, reconstituted into lipid bilayers, was used to ensure complete coupling and 9-fluorenylmethoxycarbonyl (Fmoc) deprotection. The attachment of Fmoc-TOAC-OH leads to strong immobilization of the spin label, whereas Fmoc deprotection dramatically mobilizes it, producing an EPR spectral peak that is completely resolved from that observed before deprotection. Similarly, coupling of the next amino acid (Ser) restores the spin label to strong immobilization, giving a peak that is completely resolved from that of the preceding step. For several subsequent steps, the effect of coupling and deprotection is similar but less dramatic. Thus, the sensitivity and resolution of EPR provides a quantitative monitor of completion at each of these critical steps in peptide synthesis. Mass spectrometry, circular dichroism, and Edman degradation were used in concert with EPR to verify the chemistry and characterize the secondary structure. In conclusion, the application of conventional analytical methods in combination with EPR offers an improved approach to optimize the accurate synthesis of TOAC spin-labeled membrane peptides.     

Structure and dynamics of the force-generating domain of myosin probed by multifrequency electron paramagnetic resonance

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X-and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V_i-bound biochemical state of myosin, which presumably mimics the S1.ADP.P_i state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V_i biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.     

Effect of pH on bovine liver catalase as determined by electron paramagnetic resonance.

Two major rhombic high-spin ferric heme signals are observed during the pH titration of bovine liver catalase. The less rhombic signal if dominant above pH 6.0 and the more rhombic signal below pH 6.0. Ethanol in high concentration enhances the relative intensity of the less rhombic signal. These data demonstrate the sensitivitiy of the ligand field to changes in catalase solvent and, furthermore, suggest that both rhombic configuration posses identical spectral and catalytic properties.     

A multifrequency electron spin resonance study of T4 lysozyme dynamics.

Electron spin resonance (ESR) spectroscopy at 250 GHz and 9 GHz is utilized to study the dynamics and local structural ordering of a nitroxide-labeled enzyme, T4 lysozyme (EC 3.2.1.17), in aqueous solution from 10 degrees C to 35 degrees C. Two separate derivatives, labeled at sites 44 and 69, were analyzed. The 250-GHz ESR spectra are well described by a microscopic ordering with macroscopic disordering (MOMD) model, which includes the influence of the tether connecting the probe to the protein. In the faster "time scale" of the 250-GHz ESR experiment, the overall rotational diffusion rate of the enzyme is too slow to significantly affect the spectrum, whereas for the 9-GHz ESR spectra, the overall rotational diffusion must be accounted for in the analysis. This is accomplished by using a slowly relaxing local structure model (SRLS) for the dynamics, wherein the tether motion and the overall motion are both included. In this way a simultaneous fit is successfully obtained for both the 250-GHz and 9-GHz ESR spectra. Two distinct motional/ordering modes of the probe are found for both lysozyme derivatives, indicating that the tether exists in two distinct conformations on the ESR time scale. The probe diffuses more rapidly about an axis perpendicular to its tether, which may result from fluctuations of the peptide backbone at the point of attachment of the spin probe.     

Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance

The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid-protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54 degrees C, for the three SC samples. Some lipid reorganization may occur in SC above 54 degrees C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier.     

Protein dynamics and monomer-monomer interactions in AntR activation by electron paramagnetic resonance and double electron-electron resonance

The Anthracis repressor (AntR) is a Mn(II)-activated DNA binding protein that is involved in the regulation of Mn(II) homeostasis in Bacillus anthracis. AntR is structurally and functionally homologous to Mn(II)-activated repressor from Bacillus subtillis (MntR). Our studies on AntR focus on metal-regulated activation of the protein. Line shape analysis of continuous wave electron paramagnetic resonance (EPR) spectra showed that metal binding resulted in a general reduction of backbone dynamics and that there were no further changes in backbone motion upon DNA binding. Double electron-electron resonance (DEER) pulsed EPR spectroscopy was used to measure distances between nitroxide spin labels strategically placed in dimeric AntR. The DEER data were analyzed assuming Gaussian distributions for discrete populations of spins. A structural model for AntR was built from homology to MntR, and the experimentally measured distances were simulated to distinguish between spin label and backbone motions. Together with the computational analysis, the DEER results for apo-AntR indicated relatively narrow conformational distributions for backbone residues at the dimer interface and near the metal binding site. No significant changes were observed on these sites in the presence of metal or DNA. On the other hand, the distribution of the conformers and the distances between the putative DNA binding helices decreased upon metal binding. These results suggest that the DNA binding region of AntR shows large amplitude backbone motions in the absence of metal, which may preclude sequence-specific binding to promoter sites. Metal binding narrows the range of conformations accessible in this region and shortens the mean distance between the DNA binding helices, probably resulting in alignment that optimizes promoter recognition and binding.     

Structural investigation of oxidized chlorosomes from green bacteria using multifrequency electron paramagnetic resonance up to 330 GHz

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c ^.+ [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316–325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a ^.+ [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973–6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.This revised version was published online in October 2005 with corrections to the Cover Date.     

Investigation of heavy-ion-induced sucrose radicals by electron paramagnetic resonance

The production of sucrose radicals with heavy-ion irradiation was investigated by an EPR (electron paramagnetic resonance) spectroscopic method. We examined the correlation between the production of sucrose radicals and the ion species, as well as LET (linear energy transfer). The spectral pattern obtained was the same for various ion species, including helium, carbon, neon, argon and iron ions. Quantitative EPR analyses showed that the production of sucrose radicals depended on both the ion species and the LET for the same dose of 50 Gy. The spin yield obtained showed a logarithmic correlation with the LET. In addition, the EPR response had a linear relationship with dose in the dose range of 5-60 Gy. Thus the present EPR results show that sucrose can be used to monitor the ionizing particle based on the radical yield.     

Lipid-protein interactions with cardiac phospholamban studied by spin-label electron spin resonance

Phospholamban is a cardiac regulatory protein that, in its monomeric form, inhibits the Ca(2+)-ATPase. Lipid-protein interactions with a synthetic variant of phospholamban, in which all cysteine residues are replaced with alanine, have been studied by spin-label electron spin resonance (ESR) in different lipid host membranes. Both the stoichiometry and selectivity of lipid interactions were determined from the two-component ESR spectra of phospholipid species spin-labeled on the 14 C atom of the sn-2 chain. The lipid stoichiometry is determined by the oligomeric state of the protein and the selectivity by the membrane disposition of the positively charged residues in the N-terminal section of the protein. In dimyristoylphosphatidylcholine (DMPC) membranes, the stoichiometry (N(b)) is 7 lipids/monomer for the full-length protein and 4 for the transmembrane section (residues 26-52). These stoichiometries correspond to the dimeric and pentameric forms, respectively. In palmitoyloleoylphosphatidylcholine, N(b) = 4 for both the whole protein and the transmembrane peptide. In negatively charged membranes of dimyristoylphosphatidylglycerol (DMPG), the lipid stoichiometry is N(b) = 10-11 per monomer for both the full-length protein and the transmembrane peptide. This stoichiometry corresponds to monomeric dispersion of the protein in the negatively charged lipid. The sequence of lipid selectivity is as follows: stearic acid > phosphatidic acid > phosphatidylserine = phosphatidylglycerol = phosphatidylcholine > phosphatidylethanolamine for both the full-length protein and the transmembrane peptide in DMPC. Absolute selectivities are, however, lower for the transmembrane peptide. A similar pattern of lipid selectivity is obtained in DMPG, but the absolute selectivities are reduced considerably. The results are discussed in terms of the integration of the regulatory species in the lipid membrane.     

Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues

Conformation detection and base dynamics of spin-labeled Z-DNA have been investigated by electron paramagnetic resonance (EPR) spectroscopy. The two synthesized and characterized probes used in this study were C(5)-nitroxide-labeled 2'-deoxycytidine 5'-triphosphates, pppDCAT and pppDCAVAT, which serve as suitable substrates for Micrococcus luteus DNA polymerase. Enzymatic incorporation of these probes into (dG-dC)n yields the EPR-active alternating copolymers (dG-dC,DCAT)n and (dG-dC,-DCAVAT)n. These polymers assume typical B- and Z-DNA conformations under respective low (0.1 M NaCl) and high (4.5 M NaCl) salt conditions, as evidenced by their UV-circular dichroism spectra. The EPR line shape of (dG-dC,DCAT)n in Z-form is unique and significantly different from the B-form EPR spectrum. A similar observation is made for (dG-dC,DCAVAT)n. Thus, the EPR line shapes of these spin-labeled DNAs are indicative of their local conformations. The EPR spectra, analyzed with a previously published motional model [Kao, S.-C., Polnaszek, C.F., Toppin, C.R., & Bobst, A.M. (1983) Biochemistry 22, 5563-5568], indicate tau perpendicular values of 4 and 7 ns for the B- and Z-forms, respectively. Therefore, the base dynamics of Z-DNA are about two times slower than in B-DNA.     

Simulation of the S2 state multiline electron paramagnetic resonance signal of photosystem II: a multifrequency approach.

The S2 state electron paramagnetic resonance (EPR) multiline signal of Photosystem II has been simulated at Q-band (35 Ghz), X-band (9 GHz) and S-band (4 GHz) frequencies. The model used for the simulation assumes that the signal arises from an essentially magnetically isolated MnIII-MnIV dimer, with a ground state electronic spin ST = 1/2. The spectra are generated from exact numerical solution of a general spin Hamiltonian containing anisotropic hyperfine and quadrupolar interactions at both Mn nuclei. The features that distinguish the multiline from the EPR spectra of model manganese dimer complexes (additional width of the spectrum (195 mT), additional peaks (22), internal "superhyperfine" structure) are plausibly explained assuming an unusual ligand geometry at both Mn nuclei, giving rise to normally forbidden transitions from quadrupole interactions as well as hyperfine anisotropy. The fitted parameters indicate that the hyperfine and quadrupole interactions arise from Mn ions in low symmetry environments, corresponding approximately to the removal of one ligand from an octahedral geometry in both cases. For a quadrupole interaction of the magnitude indicated here to be present, the MnIII ion must be 5-coordinate and the MnIV 5-coordinate or possibly have a sixth, weakly bound ligand. The hyperfine parameters indicate a quasi-axial anisotropy at MnIII, which while consistent with Jahn-Teller distortion as expected for a d4 ion, corresponds here to the unpaired spin being in the ligand deficient, z direction of the molecular reference axis. The fitted parameters for MnIV are very unusual, showing a high degree of anisotropy not expected in a d3 ion. This degree of anisotropy could be qualitatively accounted for by a histidine ligand providing pi backbonding into the metal dxy orbital, together with a weakly bound or absent ligand in the x direction.     

Siderophore iron transport followed by electron paramagnetic resonance spectroscopy

Siderophore iron transport was followed in Ustilago sphaerogena using isotope transport assays coupled with EPR spectroscopy. EPR spectroscopy was used as a quantitative tool to follow the rate of reduction of siderophore iron(III) to iron(II) in the cell suspension by following the disappearance of the signal at g = 4.3. This rate was compared with the rate of iron transport, measured by the disappearance of radioactively labeled iron from the medium. The transport of three iron chelates was examined: the ferric siderophores ferrichrome and ferichrome A, and iron(III) chelated to excess citrate. For the transport of ferrichrome, an iron(III) ionophore, the rate of reduction of iron(III) to iron(II) was significantly lower than the rate of uptake of isotope from the medium supernatant, which is consistent with the established mechanism of uptake of the entire complex followed by intracellular reduction to remove the iron from the ligand. However, the rate of reduction of ferrichrome A, a non-ionophore, was identical with the rate of transport of iron into the cell. Iron(III) citrate was reduced at a rate slightly lower than the rate of transport. These data suggest that reduction of iron(III) is involved in the transport of iron from ferichrome A and possibly from iron(III) citrate.     

Probing the helical tilt and dynamic properties of membrane-bound phospholamban in magnetically aligned bicelles using electron paramagnetic resonance spectroscopy

Wild-type phospholamban (WT-PLB), a Ca(2+)-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca(2+)-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13°±4° in DMPC/DHPC bicelles. TOAC spin labels placed on the WT-PLB transmembrane domain showed highly restricted motion with more than 100ns rotational correlation time (τ(c)); whereas the loop, and the cytoplasmic regions each consists of two distinct motional dynamics: one fast component in the sub-nanosecond scale and the other component is slower dynamics in the nanosecond range.     

Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data.

For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar splittings can be observed in continuous wave electron paramagnetic resonance (CW-EPR) spectra for interelectron distances as large as 30 A using perdeuterated probes. In this work, algorithms are developed for calculating CW-EPR spectra of immobilized, dipolar coupled nitroxides, and then used to define the limits of sensitivity to the interelectron distance as a function of geometry and microwave frequency. Secondly, the CW-EPR spectra of N epsilon-spin-labeled coenzyme NAD+ bound to microcrystalline, tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, and 94 GHz. These data have been analyzed, using a combination of simulated annealing and global analysis, to obtain a unique fit to the data. The values of the intermitroxide distance and the five angles defining the relative orientation of the two nitroxides are in reasonable agreement with a molecular model built from the known crystal structure. Finally, the effect of rigid body isotropic rotational diffusion on the CW-EPR spectra of dipolar coupled nitroxides has been investigated using an algorithm based on Brownian dynamics trajectories. These calculations demonstrate the sensitivity of CW-EPR spectra to dipolar coupling in the presence of rigid body rotational diffusion.     

Isothermal annealing kinetics of X-irradiated pyrene by electron paramagnetic resonance

The annealing behavior of X-irradiated stable free radicals found in pyrene (C16H10) single crystals was studied by electron paramagnetic resonance. Two processes of thermal decay kinetics were found, both with the same activation energy: 1.9 +/- 0.1 eV.     

The "beta-clasp" model of apolipoprotein A-I -- a lipid-free solution structure determined by electron paramagnetic resonance spectroscopy

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free/lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Simulation of nitroxide electron paramagnetic resonance spectra from brownian trajectories and molecular dynamics simulations

A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.     

Determination of the degree of collagen coiling by electron paramagnetic resonance

It has been shown that kinetics of the death of free radicals UV-induced at 77 degrees K in collagen is determined by two reactions having different rates. Such shape of the kinetic curve is substantiated by the spatial structure of macromolecules and permits to find easily the portion of peptide chains in the helical form and the portion of end peptides not incorporated in this structure. The degree of helical pattern of collagen from rat skin was shown to be 92%.