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Witold K. Subczynski - One of the best experts on this subject based on the ideXlab platform.
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Saturation Recovery EPR spin-labeling method for quantification of lipids in biological membrane domains.
Applied Magnetic Resonance, 2017Co-Authors: Laxman Mainali, James S. Hyde, Theodore G. Camenisch, Witold K. SubczynskiAbstract:The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and Saturation Recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [M. Raguz, L. Mainali, W. J. O'Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179-186]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student's t-test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant (P ≤ 0.05) and can be attributed to sources other than preparation/technique.
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Saturation-Recovery EPR Spin-Labeling Method for Quantification of Lipids in Domains of Biological Membranes
Biophysical Journal, 2017Co-Authors: Laxman Mainali, Witold K. SubczynskiAbstract:The presence of integral membrane proteins in biological membranes induces the formation of distinct domains in the lipid bilayer portion of these membranes. We were able to discriminate the existence of three lipid environments in intact fiber cell plasma membranes of the eye lens, namely bulk, boundary, and trapped lipids. However, our qualitative approach did not allow quantification of lipids in each domain. Recently, based on the continuous wave (CW) electron paramagnetic resonance (EPR) spectra of phospholipid analog and cholesterol analog spin labels, we developed a method that allows evaluation of the relative amount of phospholipids and cholesterol in the bulk lipid domain and in the boundary plus trapped lipid domain [M. Raguz, L. Mainali,W. J. O’Brien and W. K. Subczynski, (2015), Exp. Eye Res., 140:179-186]. Here, we will present a new approach that, based on Saturation-Recovery (SR) EPR, allows evaluation of the relative amount of phospholipids and cholesterol in the bulk plus boundary lipid domain and in the trapped lipid domain. Spectrometer conditions for the successful application of this method to quantify lipids in membrane domains are described. These two methods clearly demonstrate that the method's time window allows the separation of results from the different domains. CW EPR mixes results from the boundary and trapped lipids, while SR EPR mixes results from the bulk and boundary lipids. These two methods complement each other, providing a more complete picture of lipid lateral organization in intact membranes. The abilities of these methods are illustrated in the intact fiber cell plasma membranes from porcine eye lenses.Acknowledgments: This work was supported by grants EY015526, EB001980, and EY001931 from the National Institutes of Health.
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Oxygen transport parameter in mei Saturation Recovery measurements relaxation times of spin labels
2016Co-Authors: Akihiro Kusumi, Witold K. Subczynski, JamesAbstract:Spin-lattice relaxation time (T1) measurements of nitroxide radical spin labels in membranes have been made by using the Saturation-Recovery technique. Stearic acid and sterol- type labels were used as probes of dimyristoylphosphatidylcholine liposomes from 0?C to 36?C. In the absence of oxygen, the range of variation of T1 over all samples and conditions is about a factor of 3. Heisenberg exchange between oxygen and spin labels is an effective T1 mechanism for the spin labels. The full range of vari- ation of T1 in the presence of air is about a factor of 100. It is sug- gested that the oxygen transport parameter W = Ti1' (air) - Ti 1 (N2) is a useful new monitor of membrane fluidity that reports on translational diffusion of small molecules. The values of W change at the prephase and main phase transitions and vary in complex ways. Arguments are advanced that the data are indic- ative of anisotropic translational diffusion of oxygen.
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Spin-label Saturation-Recovery EPR at W-band: applications to eye lens lipid membranes.
Journal of Magnetic Resonance, 2011Co-Authors: Laxman Mainali, James S. Hyde, Theodore G. Camenisch, Marija Raguz, Witold K. SubczynskiAbstract:Abstract Saturation-Recovery (SR) EPR at W-band (94 GHz) to obtain profiles of the membrane fluidity and profiles of the oxygen transport parameter is demonstrated for lens lipid membranes using phosphatidylcholine (n-PC), stearic acid (n-SASL), and cholesterol analog (ASL and CSL) spin labels, and compared with results obtained in parallel experiments at X-band (9.4 GHz). Membranes were derived from the total lipids extracted from 2-year-old porcine lens cortex and nucleus. Two findings are especially significant. First, measurements of the spin–lattice relaxation times T1 for n-PCs allowed T1 profiles across the membrane to be obtained. These profiles reflect local membrane properties differently than profiles of the order parameter. Profiles obtained at W-band are, however, shifted to longer T1 values compared to those obtained at X-band. Second, using cholesterol analog spin labels and relaxation agents (hydrophobic oxygen and water-soluble NiEDDA), the cholesterol bilayer domain was discriminated in membranes made from lipids of the lens nucleus. However, membranes made from cortical lipids show a single homogeneous environment. Profiles of the oxygen transport parameter obtained from W-band measurements are practically identical to those obtained from X-band measurements, and are very similar to those obtained earlier at X-band for membranes made of 2-year-old bovine cortical and nuclear lens lipids (M. Raguz, J. Widomska, J. Dillon, E.R. Gaillard, W.K. Subczynski, Biochim. Biophys. Acta 1788 (2009) 2380–2388). Results demonstrate that SR EPR at W-band has the potential to be a powerful tool for studying samples of small volume, ∼30 nL, compared with the sample volume of ∼3 μL at X-band.
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Saturation Recovery EPR and ELDOR at W-band for spin labels
Journal of Magnetic Resonance, 2008Co-Authors: Wojciech Froncisz, Theodore G. Camenisch, Joseph J. Ratke, James R. Anderson, Witold K. Subczynski, Robert A. Strangeway, Jason W. Sidabras, James S. HydeAbstract:Abstract 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 TE 011 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 T 1 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.
Wojciech Froncisz - One of the best experts on this subject based on the ideXlab platform.
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Saturation Recovery EPR and ELDOR at W-band for spin labels
Journal of Magnetic Resonance, 2008Co-Authors: Wojciech Froncisz, Theodore G. Camenisch, Joseph J. Ratke, James R. Anderson, Witold K. Subczynski, Robert A. Strangeway, Jason W. Sidabras, James S. HydeAbstract:Abstract 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 TE 011 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 T 1 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.
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Accessibility and Dynamics of Nitroxide Side Chains in T4 Lysozyme Measured by Saturation Recovery EPR
Biophysical Journal, 2005Co-Authors: Janusz Pyka, Jan Ilnicki, Christian Altenbach, Wayne L. Hubbell, Wojciech FronciszAbstract:Long pulse Saturation Recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential Saturation Recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O2 or NiEDDA, global analysis of the Saturation Recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power Saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128–135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the Saturation Recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the Saturation Recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.
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spin label epr t1 values using Saturation Recovery from 2 to 35 ghz
Journal of Physical Chemistry B, 2004Co-Authors: James S. Hyde, Jun-jie Yin, Theodore G. Camenisch, Joseph J. Ratke, Witold K. Subczynski, Wojciech FronciszAbstract:EPR Saturation-Recovery (SR) measurements of the electron spin-lattice relaxation time, T 1, of nitroxideradical spin probes have been made from 2 to 35 GHz. T1 values of small water-soluble spin probes increase linearly with microwave frequency throughout the full range of available frequencies. T1 values of four commonly used hydrophobic probes in lipid bilayers also increase with frequency, but the dependence is weaker and complex. Contributions of dissolved molecular oxygen to relaxation rates were independent of microwave frequency. T1 values of 15 N-containing labels are always somewhat longer than for 14 N labels. Details of the Q-band SR spectrometer, which is based on frequency translation technology, are provided. A new way to suppress free induction decay signals in SR experiments has been found: pump and observing frequencies time-locked and separated by about 1 kHz in frequency. A novel three-loop-two-gap resonator with a sample volume of 30 nl was used for the Q-band measurements. It is concluded that Q-band is a favorable frequency for SR spin-label oximetry studies.
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Spin-Label EPR T1 Values Using Saturation Recovery from 2 to 35 GHz†
The Journal of Physical Chemistry B, 2004Co-Authors: James S. Hyde, Jun-jie Yin, Theodore G. Camenisch, Joseph J. Ratke, Witold K. Subczynski, Wojciech FronciszAbstract:EPR Saturation-Recovery (SR) measurements of the electron spin-lattice relaxation time, T1, of nitroxide-radical spin probes have been made from 2 to 35 GHz. T1 values of small water-soluble spin probes increase linearly with microwave frequency throughout the full range of available frequencies. T1 values of four commonly used hydrophobic probes in lipid bilayers also increase with frequency, but the dependence is weaker and complex. Contributions of dissolved molecular oxygen to relaxation rates were independent of microwave frequency. T1 values of 15N-containing labels are always somewhat longer than for 14N labels. Details of the Q-band SR spectrometer, which is based on frequency translation technology, are provided. A new way to suppress free induction decay signals in SR experiments has been found: pump and observing frequencies time-locked and separated by about 1 kHz in frequency. A novel three-loop−two-gap resonator with a sample volume of 30 nl was used for the Q-band measurements. It is concl...
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Pulse Saturation Recovery, pulse ELDOR, and free induction decay electron paramagnetic resonance detection using time-locked subsampling
Review of Scientific Instruments, 2001Co-Authors: Wojciech Froncisz, Theodore G. Camenisch, Joseph J. Ratke, James S. HydeAbstract:Time locked subsampling (TLSS) in electron paramagnetic resonance (EPR) involves the steps of (i) translation of the signal from a microwave carrier to an intermediate frequency (IF) carrier where the (IF) offset between the signal oscillator and local oscillator frequencies is synthesized, (ii) sampling the IF carrier four times in an odd number of cycles, say 4 in 3, where the analog-to-digital (A/D) converter is driven by a frequency synthesizer that has the same clock input as the IF synthesizer, (iii) signal averaging as required for adequate signal to noise, (iv) separating the even and odd digitized words into two separate signal channels, which correspond to signals in phase and in quadrature with respect to the IF carrier, i.e., I and Q, and (v) detecting the envelope of I and also of Q by changing the signs of alternate words in each of the two channels. TLSS detection has been demonstrated in three forms of pulse EPR spectroscopy at X band: Saturation Recovery, pulse electron–electron double resonance, and free induction decay. The IF was 187.5 MHz, the A/D converter frequency was 250 MHz, the overall bandwidth was 125 MHz, and the bandwidths for the separate I and Q channels were each 62.5 MHz. Experiments were conducted on nitroxide radical spin labels. The work was directed towards development of methodology to monitor bimolecular collisions of oxygen with spin labels in a context of site-directed spin labeling.
Bruce H. Robinson - One of the best experts on this subject based on the ideXlab platform.
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explanation of spin lattice relaxation rates of spin labels obtained with multifrequency Saturation Recovery epr
Journal of Physical Chemistry A, 2005Co-Authors: Colin Mailer, And Robert D. Nielsen, Bruce H. RobinsonAbstract:Electron paramagnetic resonance (EPR) pulsed Saturation Recovery (pSR) measurements of spin−lattice relaxation rates have been made on nitroxide-containing fatty acids embedded in lipid bilayers by Hyde and co-workers. The data have been collected for a number of spin-labeled fatty acids at several microwave spectrometer frequencies (from 2 to 35 GHz). We compare these spin−lattice relaxation rates to those predicted by the Redfield theory incorporating several mechanisms. The dominant relaxation mechanism at low spectrometer frequencies is the electron−nuclear dipolar (END) process, with spin rotation (SR), chemical shift anisotropy (CSA), and a generalized spin diffusion (GSD) mechanism all contributing. The use of a wide range of spectrometer frequencies makes clear that the dynamics cannot be modeled adequately by rigid-body isotropic rotational motion. The dynamics of rigid-body anisotropic rotational motion is sufficient to explain the experimental relaxation rates within the experimental error. Mor...
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Explanation of Spin−Lattice Relaxation Rates of Spin Labels Obtained with Multifrequency Saturation Recovery EPR
The Journal of Physical Chemistry A, 2005Co-Authors: Colin Mailer, And Robert D. Nielsen, Bruce H. RobinsonAbstract:Electron paramagnetic resonance (EPR) pulsed Saturation Recovery (pSR) measurements of spin−lattice relaxation rates have been made on nitroxide-containing fatty acids embedded in lipid bilayers by Hyde and co-workers. The data have been collected for a number of spin-labeled fatty acids at several microwave spectrometer frequencies (from 2 to 35 GHz). We compare these spin−lattice relaxation rates to those predicted by the Redfield theory incorporating several mechanisms. The dominant relaxation mechanism at low spectrometer frequencies is the electron−nuclear dipolar (END) process, with spin rotation (SR), chemical shift anisotropy (CSA), and a generalized spin diffusion (GSD) mechanism all contributing. The use of a wide range of spectrometer frequencies makes clear that the dynamics cannot be modeled adequately by rigid-body isotropic rotational motion. The dynamics of rigid-body anisotropic rotational motion is sufficient to explain the experimental relaxation rates within the experimental error. Mor...
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Comparing continuous wave progressive Saturation EPR and time domain Saturation Recovery EPR over the entire motional range of nitroxide spin labels.
Journal of Magnetic Resonance, 2004Co-Authors: Robert D. Nielsen, Colin Mailer, Stéphane Canaan, James Gladden, Michael H. Gelb, Bruce H. RobinsonAbstract:The measurement of spin–lattice relaxation rates from spin labels, such as nitroxides, in the presence and absence of spin relaxants provides information that is useful for determining biomolecular properties such as nucleic acid dynamics and the interaction of proteins with membranes. We compare X-band continuous wave (CW) and pulsed or time domain (TD) EPR methods for obtaining spin–lattice relaxation rates of spin labels across the entire range of rotational motion to which relaxation rates are sensitive. Model nitroxides and spin-labeled biological species are used to illustrate the potential complications that arise in extracting relaxation data under conditions typical to biological experiments. The effect of super hyperfine (SHF) structure is investigated for both CW and TD spectra. First and second harmonic absorption and dispersion CW spectra of the nitroxide spin label, TEMPOL, are all fit simultaneously to a model of SHF structure over a range of microwave amplitudes. The CW spectra are novel because all harmonics and microwave phases were acquired simultaneously using our homebuilt CW/TD spectrometer. The effect of the SHF structure on the pulsed free induction decay (FID) and pulsed Saturation Recovery spectrum is shown for both protonated and deuterated TEMPOL. We present novel pulsed Saturation Recovery measurements on biological molecules, including spin–lattice relaxation rates of spin-labeled proteins and spin-labeled double-stranded DNA. The impact of structure and dynamics on relaxation rates are discussed in the context of each of these examples. Collisional relaxation rates with oxygen and transition metal paramagnetic relaxants are extracted using both continuous wave and time domain methods. The extent of the errors inherent in the CW method and the advantages of pulsed methods for unambiguously measuring collisional relaxation rates are discussed. Spin–lattice relaxation rates, determined by both CW and pulsed methods, are used to determine the electrostatic potential on the surface of a protein.
James S. Hyde - One of the best experts on this subject based on the ideXlab platform.
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Saturation Recovery EPR spin-labeling method for quantification of lipids in biological membrane domains.
Applied Magnetic Resonance, 2017Co-Authors: Laxman Mainali, James S. Hyde, Theodore G. Camenisch, Witold K. SubczynskiAbstract:The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and Saturation Recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [M. Raguz, L. Mainali, W. J. O'Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179-186]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student's t-test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant (P ≤ 0.05) and can be attributed to sources other than preparation/technique.
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Spin-label Saturation-Recovery EPR at W-band: applications to eye lens lipid membranes.
Journal of Magnetic Resonance, 2011Co-Authors: Laxman Mainali, James S. Hyde, Theodore G. Camenisch, Marija Raguz, Witold K. SubczynskiAbstract:Abstract Saturation-Recovery (SR) EPR at W-band (94 GHz) to obtain profiles of the membrane fluidity and profiles of the oxygen transport parameter is demonstrated for lens lipid membranes using phosphatidylcholine (n-PC), stearic acid (n-SASL), and cholesterol analog (ASL and CSL) spin labels, and compared with results obtained in parallel experiments at X-band (9.4 GHz). Membranes were derived from the total lipids extracted from 2-year-old porcine lens cortex and nucleus. Two findings are especially significant. First, measurements of the spin–lattice relaxation times T1 for n-PCs allowed T1 profiles across the membrane to be obtained. These profiles reflect local membrane properties differently than profiles of the order parameter. Profiles obtained at W-band are, however, shifted to longer T1 values compared to those obtained at X-band. Second, using cholesterol analog spin labels and relaxation agents (hydrophobic oxygen and water-soluble NiEDDA), the cholesterol bilayer domain was discriminated in membranes made from lipids of the lens nucleus. However, membranes made from cortical lipids show a single homogeneous environment. Profiles of the oxygen transport parameter obtained from W-band measurements are practically identical to those obtained from X-band measurements, and are very similar to those obtained earlier at X-band for membranes made of 2-year-old bovine cortical and nuclear lens lipids (M. Raguz, J. Widomska, J. Dillon, E.R. Gaillard, W.K. Subczynski, Biochim. Biophys. Acta 1788 (2009) 2380–2388). Results demonstrate that SR EPR at W-band has the potential to be a powerful tool for studying samples of small volume, ∼30 nL, compared with the sample volume of ∼3 μL at X-band.
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Saturation Recovery EPR and ELDOR at W-band for spin labels
Journal of Magnetic Resonance, 2008Co-Authors: Wojciech Froncisz, Theodore G. Camenisch, Joseph J. Ratke, James R. Anderson, Witold K. Subczynski, Robert A. Strangeway, Jason W. Sidabras, James S. HydeAbstract:Abstract 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 TE 011 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 T 1 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.
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spin label epr t1 values using Saturation Recovery from 2 to 35 ghz
Journal of Physical Chemistry B, 2004Co-Authors: James S. Hyde, Jun-jie Yin, Theodore G. Camenisch, Joseph J. Ratke, Witold K. Subczynski, Wojciech FronciszAbstract:EPR Saturation-Recovery (SR) measurements of the electron spin-lattice relaxation time, T 1, of nitroxideradical spin probes have been made from 2 to 35 GHz. T1 values of small water-soluble spin probes increase linearly with microwave frequency throughout the full range of available frequencies. T1 values of four commonly used hydrophobic probes in lipid bilayers also increase with frequency, but the dependence is weaker and complex. Contributions of dissolved molecular oxygen to relaxation rates were independent of microwave frequency. T1 values of 15 N-containing labels are always somewhat longer than for 14 N labels. Details of the Q-band SR spectrometer, which is based on frequency translation technology, are provided. A new way to suppress free induction decay signals in SR experiments has been found: pump and observing frequencies time-locked and separated by about 1 kHz in frequency. A novel three-loop-two-gap resonator with a sample volume of 30 nl was used for the Q-band measurements. It is concluded that Q-band is a favorable frequency for SR spin-label oximetry studies.
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Spin-Label EPR T1 Values Using Saturation Recovery from 2 to 35 GHz†
The Journal of Physical Chemistry B, 2004Co-Authors: James S. Hyde, Jun-jie Yin, Theodore G. Camenisch, Joseph J. Ratke, Witold K. Subczynski, Wojciech FronciszAbstract:EPR Saturation-Recovery (SR) measurements of the electron spin-lattice relaxation time, T1, of nitroxide-radical spin probes have been made from 2 to 35 GHz. T1 values of small water-soluble spin probes increase linearly with microwave frequency throughout the full range of available frequencies. T1 values of four commonly used hydrophobic probes in lipid bilayers also increase with frequency, but the dependence is weaker and complex. Contributions of dissolved molecular oxygen to relaxation rates were independent of microwave frequency. T1 values of 15N-containing labels are always somewhat longer than for 14N labels. Details of the Q-band SR spectrometer, which is based on frequency translation technology, are provided. A new way to suppress free induction decay signals in SR experiments has been found: pump and observing frequencies time-locked and separated by about 1 kHz in frequency. A novel three-loop−two-gap resonator with a sample volume of 30 nl was used for the Q-band measurements. It is concl...
Wayne L. Hubbell - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Saturation Recovery Amplitudes to Characterize Conformational Exchange in Spin-Labeled Proteins
Applied Magnetic Resonance, 2017Co-Authors: Michael D. Bridges, Christian Altenbach, Zhongyu Yang, Wayne L. HubbellAbstract:Analysis of Saturation Recovery data from spin-labeled proteins is extended to include the amplitudes in addition to the Recovery rates for two-site exchange. It is shown that the Recovery amplitudes depend strongly on the exchange rate between states as well as their populations and this dependence provides a simple criterion to identify exchange rates in the 10–1000 kHz range. Analysis of experimental SR relaxation curves via the uniform penalty (UPEN) method allows for reliable identification of single, double, or other multiple-component traces, and global fitting of a set of relaxation curves using both relaxation rates and amplitudes determined from the UPEN fits allows for the estimation of exchange rate in the above domain. The theory is tested on simple model systems, and applied to the determination of conformational exchange rates in spin-labeled mutants of T4 Lysozyme and intestinal fatty acid binding protein. Finally, an example of T 1-weighted spectral editing is provided for systems in the slow exchange limit.
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Saturation Recovery EPR and Nitroxide Spin Labeling for Exploring Structure and Dynamics in Proteins.
Methods in Enzymology, 2015Co-Authors: Zhongyu Yang, Christian Altenbach, Michael D. Bridges, Michael T. Lerch, Wayne L. HubbellAbstract:Experimental techniques capable of determining the structure and dynamics of proteins are continuously being developed in order to understand protein function. Among existing methods, site-directed spin labeling in combination with Saturation Recovery (SR) electron paramagnetic resonance spectroscopy contributes uniquely to the determination of secondary and tertiary protein structure under physiological conditions, independent of molecular weight and complexity. In addition, SR of spin labeled proteins was recently demonstrated to be sensitive to conformational exchange events with characteristic lifetimes on the order of μs, a time domain that presents a significant challenge to other spectroscopic techniques. In this chapter, we present the theoretical background necessary to understand the capabilities of SR as applied to spin labeled proteins, the instrumental requirements, and practical experimental considerations necessary to obtain interpretable data, and the use of SR to obtain information on protein: (1) secondary structure via solvent accessibility measurements, (2) tertiary structure using interspin distance measurements, and (3) conformational exchange.
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Long-range distance measurements in proteins at physiological temperatures using Saturation Recovery EPR spectroscopy.
Journal of the American Chemical Society, 2014Co-Authors: Zhongyu Yang, Michael D. Bridges, Gonzalo Jiménez-osés, Carlos J. López, Kendall N. Houk, Wayne L. HubbellAbstract:Site-directed spin labeling in combination with EPR is a powerful method for providing distances on the nm scale in biological systems. The most popular strategy, double electron-electron resonance (DEER), is carried out at cryogenic temperatures (50-80 K) to increase the short spin-spin relaxation time (T2) upon which the technique relies. A challenge is to measure long-range distances (20-60 A) in proteins near physiological temperatures. Toward this goal we are investigating an alternative approach based on the distance-dependent enhancement of spin-lattice relaxation rate (T1(-1)) of a nitroxide spin label by a paramagnetic metal. With a commonly used nitroxide side chain (R1) and Cu(2+), it has been found that interspin distances ≤25 A can be determined in this way (Jun et al. Biochemistry 2006, 45, 11666). Here, the upper limit of the accessible distance is extended to ≈40 A using spin labels with long T1, a high-affinity 5-residue Cu(2+) binding loop inserted into the protein sequence, and pulsed Saturation Recovery to measure relaxation enhancement. Time-domain Cu(2+) electron paramagnetic resonance, quantum mechanical calculations, and molecular dynamics simulations provide information on the structure and geometry of the Cu(2+) loop and indicate that the metal ion is well-localized in the protein. An important aspect of these studies is that both Cu(2+)/nitroxide DEER at cryogenic temperatures and T1 relaxation measurements at room temperature can be carried out on the same sample, allowing both validation of the relaxation method and assessment of the effect of freezing on protein structure.
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Resolving Conformational and Rotameric Exchange in Spin-Labeled Proteins Using Saturation Recovery EPR.
Applied Magnetic Resonance, 2009Co-Authors: Michael D. Bridges, Kálmán Hideg, Wayne L. HubbellAbstract:The function of many proteins involves equilibria between conformational substates, and to elucidate mechanisms of function it is essential to have experimental tools to detect the presence of conformational substates and to determine the time scale of exchange between them. Site-directed spin labeling (SDSL) has the potential to serve this purpose. In proteins containing a nitroxide side chain (R1), multicomponent electron paramagnetic resonance (EPR) spectra can arise either from equilibria involving different conformational substates or rotamers of R1. To employ SDSL to uniquely identify conformational equilibria, it is thus essential to distinguish between these origins of multicomponent spectra. Here we show that this is possible based on the time scale for exchange of the nitroxide between distinct environments that give rise to multicomponent EPR spectra; rotamer exchange for R1 lies in the ≈0.1-1 μs range, while conformational exchange is at least an order of magnitude slower. The time scales of exchange events are determined by Saturation Recovery EPR, and in favorable cases, the exchange rate constants between substates with lifetimes of approximately 1-70 μs can be estimated by the approach.
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Accessibility and Dynamics of Nitroxide Side Chains in T4 Lysozyme Measured by Saturation Recovery EPR
Biophysical Journal, 2005Co-Authors: Janusz Pyka, Jan Ilnicki, Christian Altenbach, Wayne L. Hubbell, Wojciech FronciszAbstract:Long pulse Saturation Recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential Saturation Recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O2 or NiEDDA, global analysis of the Saturation Recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power Saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128–135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the Saturation Recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the Saturation Recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.
