Rotational dynamics of phospholamban determined by multifrequency electron paramagnetic resonance

We have used multifrequency electron paramagnetic resonance to define the multistate structural dynamics of an integral membrane protein, phospholamban (PLB), in a lipid bilayer. PLB is a key regulator of cardiac calcium transport, and its function requires transitions between distinct states of intramolecular dynamics. Monomeric PLB was synthesized with the TOAC spin label at positions 11 (in the cytoplasmic domain) and 46 (in the transmembrane domain) and reconstituted into lipid bilayers. Unlike other protein spin labels, TOAC reports directly the motion of the peptide backbone, so quantitative analysis of its dynamics is worthwhile. Electron paramagnetic resonance spectra at 9.4 GHz (X-band) and 94 GHz (W-band) were analyzed in terms of anisotropic rotational diffusion of the two domains. Motion of the transmembrane domain is highly restricted, while the cytoplasmic domain exhibits two distinct conformations, a major one with moderately restricted nanosecond dynamics (T) and another with nearly unrestricted subnanosecond motion (R). The global analysis of spectra at two frequencies yielded values for the rotational correlation times and order parameters that were much more precisely determined than at either frequency alone. Multifrequency EPR is a powerful approach for analysis of complex rotational dynamics of proteins.     

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.     

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.     

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.     

Protected peptide p-nitroanilides by solid-phase synthesis

Summary Peptidep-nitroanilides (peptidepNAs) have found wide application as chromogenic substrates. An improved SPPS method to synthesize rapidly and in good yield a broad range of peptidepNAs under mild conditions will be presented here. To obtain a suitable carrier, the (4-aminophenyl)aminocarbonyloxy derivatives of Wang resin and Sasrin were synthesized. SPPS employing Fmoc/tBu-based protection followed by acidolytic cleavage yielded the (protected) peptidep-aminoanilide which was oxidized with sodium perborate in acetic acid to yield the (protected) peptidepNA. Side-chain protection proved to be advantageous. Acid-labile peptidepNAs such as the proteasome substrate Z-Glu(OtBu)-Ala-Leu-pNA thus can be obtained directly. The behavior of Cys, Met, Tyr and Trp being susceptible towards oxidation was studied more closely. A preliminary account of this work was presented at the 8th Int. Conf. Polymer-Based Technology, Ma'ale Hachamisha, Israel, June 28–July 3, 1998     

Protected peptide p-nitroanilides by solid-phase synthesis

Peptide p-nitroanilides (peptide p> NAs) have found wide application as chromogenic substrates. An improved SPPS method to synthesize rapidly and in good yield a broad range of peptide pNAs under mild conditions will be presented here. To obtain a suitable carrier, the (4-aminophenyl)aminocarbonyloxy derivatives of Wang resin and Sasrin were synthesized. SPPS employing Fmoc/tBu-based protection followed by acidolytic cleavage yielded the (protected) peptide p-aminoanilide which was oxidized with sodium perborate in acetic acid to yield the(protected) peptide pNA. Side-chain protectionproved to be advantageous. Acid-labile peptide pNAs such as the proteasome substrate Z-Glu(OtBu)-Ala-Leu-pNA thus can be obtaineddirectly. The behavior of Cys, Met, Tyr and Trp being susceptible towards oxidation was studied more closely.     

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.     

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.     

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: electron paramagnetic resonance, circular dichroism, and fluorescence studies

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.     

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.     

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.     

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.     

Substrate binding to DNA photolyase studied by electron paramagnetic resonance spectroscopy.

Structural changes in Escherichia coli DNA photolyase induced by binding of a (cis,syn)-cyclobutane pyrimidine dimer (CPD) are studied by continuous-wave electron paramagnetic resonance and electron-nuclear double resonance spectroscopies, using the flavin adenine dinucleotide (FAD) cofactor in its neutral radical form as a naturally occurring electron spin probe. The electron paramagnetic resonance/electron-nuclear double resonance spectral changes are consistent with a large distance (> or =0.6 nm) between the CPD lesion and the 7,8-dimethyl isoalloxazine ring of FAD, as was predicted by recent model calculations on photolyase enzyme-substrate complexes. Small shifts of the isotropic proton hyperfine coupling constants within the FAD's isoalloxazine moiety can be understood in terms of the cofactor binding site becoming more nonpolar because of the displacement of water molecules upon CPD docking to the enzyme. Molecular orbital calculations of hyperfine couplings using density functional theory, in conjunction with an isodensity polarized continuum model, are presented to rationalize these shifts in terms of the changed polarity of the medium surrounding the FAD cofactor.     

Backbone folding of the polypeptide cardiac stimulant anthopleurin-A determined by nuclear magnetic resonance, distance geometry and molecular dynamics

The solution conformation of the cardiac stimulatory sea anemone polypeptide anthopleurin-A has been characterised using distance geometry and restrained molecular dynamics calculations. A set of 253 approximate interproton distance restraints and 14 peptide backbone torsion angle restraints derived from two-dimensional 1H-NMR spectra at 500 MHz were used as input for these calculations. 13 structures generated by either metric matrix or variable target function distance geometry calculations were refined using energy minimisation and restrained molecular dynamics. The resulting structures contain a region of twisted antiparellel beta-sheet to which two separate regions of unordered chain are linked by three disulphide bonds. Two loops, one including Pro-41 and the other encompassing residues 10-18, are poorly defined by the NOE data.     

Applications of electron paramagnetic resonance to studies of neurological disease

Electron paramagnetic resonance spectroscopy (EPR) has the potential to give much detail on the structure of the paramagnetic transition ion coordination sites, principally of Cu²⁺, in a number of proteins associated with central nervous system diseases. Since these sites have been implicated in misfolding/mis-oligomerisation events associated with neurotoxic molecular species and/or the catalysis of damaging redox reactions in neurodegeneration, an understanding of their structure is important to the development of therapeutic agents. Nevertheless EPR, by its nature an in vitro technique, has its limitations in the study of such complex biochemical systems involving self-associating proteins that are sensitive to their chemical environment. These limitations are at the instrumental and theoretical level, which must be understood and the EPR data interpreted in the light of other biophysical and biochemical studies if useful conclusions are to be drawn. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience, held in Melbourne on 11 July 2007.     

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.