A Spirocyclohexyl Nitroxide Amino Acid Spin Label for Pulsed EPR Distance Measurements (original) (raw)
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A Spirocyclohexyl Nitroxide Amino Acid Spin Label for Pulsed EPR Spectroscopy Distance Measurements
Chemistry - A European Journal, 2010
Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide α-amino acid and its N-(9fluorenylmethoxycarbonyl) (Fmoc) derivative, has been synthesized that exhibit slow enough spin echo dephasing to permit accurate distance measurements by pulse EPR at temperatures up to 125 K in 1:1 water:glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR to the study of structure and conformation for peptides and proteins.
A Spirocyclohexyl Nitroxide Amino Acid Spin Label for Pulsed EPR Spectroscopy Distance Measurements
Chemistry: A European Journal, 2010
Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide α-amino acid and its N-(9fluorenylmethoxycarbonyl) (Fmoc) derivative, has been synthesized that exhibit slow enough spin echo dephasing to permit accurate distance measurements by pulse EPR at temperatures up to 125 K in 1:1 water:glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR to the study of structure and conformation for peptides and proteins.
Pulsed EPR Distance Measurements in Soluble Proteins by Site-Directed Spin Labeling (SDSL)
Current Protocols in Protein Science, 2001
The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electronelectron resonance (DEER) technique. This unit focuses on EPR-based distance measurements by site-directed spin labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV-1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance-distribution profiles. Experimental considerations, sample preparation, and error analysis for artifact suppression are also outlined herein.
Pulsed electron-electron double resonance (PELDOR) coupled with site-directed spin labeling is a powerful technique for the elucidation of protein or nucleic acid, macromolecular structure and interactions. The intrinsic high sensitivity of electron paramagnetic resonance enables measurement on small quantities of biomacromolecules, however short relaxation times impose a limit on the sensitivity and size of distances that can be measured using this technique. The persistence of the electron spin-echo, in the PELDOR experiment, is one of the most crucial limitations to distance measurement. At a temperature of around 50 K one of the predominant factors affecting persistence of an echo, and as such, the sensitivity and measurable distance between spin labels, is the electron spin echo dephasing time (T m). It has become normal practice to use deuterated solvents to extend T m and recently it has been demonstrated that deuteration of the underlying protein significantly extends T m. Here we examine the spatial effect of segmental deuteration of the underlying protein, and also explore the concentration and temperature dependence of highly deuterated systems.
Structural studies on membrane proteins using non-linear spin label EPR spectroscopy
Cellular & molecular biology letters, 2002
Non-linear electron spin resonance (EPR) techniques suitable for measuring proximity relationships in membranes are reviewed. These were developed during the past decade in order to measure changes sensitively in the spin-lattice relaxation time (T1) of nitroxyl spin labels covalently attached to membrane lipids or proteins. In combination with paramagnetic quenching agents and double spin-labelling, the methods were further developed for distance measurements. Selected examples are given to illustrate different methods, and types of data obtained for both integral and peripheral membrane proteins.
Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR
Biophysical journal, 2015
Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.
Spectroscopy-an International Journal, 2010
Investigations on the structure and function of biomolecules often depend on the availability of topological information to build up structural models or to characterize conformational changes during function. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) allow to determine intra-and intermolecular distances in the range from 4-70 Å, covering the range of interest for biomolecules. The approach does not require crystalline samples and is well suited also for molecules exhibiting intrinsic flexibility. This article is intended to give an overview on pulsed EPR in conjunction with SDSL to study protein interactions as well as conformational changes, exemplified on the tRNA modifying enzyme MnmE.
Synthesis and Measurements. 1.a General procedures and materials. Throughout the following paragraphs labels "ZY241" and alike correspond to sample or experiment codes directly traceable to the laboratory notebooks or raw data. All reactions were carried under N2 atmosphere with anhydrous solvents. Tetrahydrofuran (THF) was freshly distilled from sodium/benzophenone prior to use or, like dichloromethane (DCM), used directly from solvent purification system (LC Technology Solutions). 1,2-Dichloroethane (1,2-DCE) was dried by 4 Å molecular sieves overnight, degassed by N2 gas flow, transferred via cannula into Schlenk vessel, which was covered with aluminum foil and stored on high vacuum line. Per-deuterated solvents for NMR spectroscopy were obtained from Cambridge Isotope Laboratories. All other commercially available chemicals were obtained from either Aldrich or Acros, unless indicated otherwise; e.g., starting material, endo-3-amino-9-methyl-9-azabicyclo[3.3.1]nonane, was obtained from AK Scientific, Inc. Standard techniques for synthesis under inert atmosphere, using vaccum lines and Schlenk glassware, were employed. Column chromatography (0-20 psig pressure) was performed by using Sorbtech neutral aluminum oxide (32-63 m) or silica gel (60 Å, 40-75 m). Analytical TLC plates were carried out on 0.25 mm MilliporeSigma silica plates (60F-254) or 0.25 mm Sorbtech neutral aluminum oxide plates (w/UV254), using UV light as the visualizing agent, or ninhydrin and heat as developing agent. NMR spectra were obtained using Bruker spectrometers (1 H, 300, 400, 600, and 700 MHz) using chloroform-d (CDCl3), water-d2, acetone-d6, or DMSO-d6, as solvent. The 700 MHz instrument was equipped with a cryoprobe. The chemical shift references were as follows: (1 H) chloroform-h, 7.26 ppm; (1 H) acetone-d5, 2.05 ppm; (13 C) acetone-d6, 30.23 ppm; (1 H) water-d1, 4.79 ppm; (1 H) DMSO-d5, 2.50 ppm; (13 C) DMSO-d6, 39.51 ppm. Typical 1D FID was subjected to exponential multiplication with an exponent of 0.1 Hz (for 1 H) and 1.0-2.0 Hz (for 13 C). IR spectra were obtained using a commercial instrument, equipped with an ATR sampling accessory. MS analyses were carried out at local facilities for mass spectrometry. S4 1.b. X-ray crystallography. Crystallographic data were deposited in the Cambridge Crystallographic Data Centre (CCDC 1916093). The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data\_request/cif. The single crystals of nitroxide 1 were obtained by slow solvent evaporation from a pentane/chloroform solvent mixture; the solvent mixture was obtained by a slow diffusion of pentane into a solution of 1 in chloroform (sample label: ZY241). The data collections for crystal of 1 were carried out using Mo Kα radiation (graphite monochromator, = 0.71073 Å) with a frame time of 20 seconds and a detector distance of 4.00 cm. A collection strategy was calculated and complete data to a resolution of 0.71 Å with a redundancy of 4 were collected. Five major sections of frames were collected with 0.50º ω and φ scans. A total of 953 frames were collected. The total exposure time was 5.34 hours. Nitroxide 1. The integration (SAINT) S1 of the data using an orthorhombic unit cell yielded a total of 20397 reflections to a maximum θ angle of 30.04° (0.71 Å resolution), of which 3216 were independent (average redundancy 6.342, completeness = 100.0%, Rint = 2.80%, Rsig = 1.87%) and 2904 (90.30%) were greater than 2σ(F2). The final cell constants of a = 8.3415(4) Å, b = 10.1215(4) Å, c = 25.9996(12) Å, volume = 2195.11(17) Å 3 , are based upon the refinement of the XYZ-centroids of 99 reflections above 20 σ(I) with 9.521° < 2θ < 58.31°. Data were corrected for absorption effects using the multi-scan method (SADABS). S2 The ratio of minimum to maximum apparent transmission was 0.854. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.6368 and 0.7460. The space group Pbca was determined based on intensity statistics and systematic absences. The structure was solved and refined using the SHELX suite of programs. S3 An intrinsic-methods solution was calculated, which provided most non-hydrogen atoms from the E-map. Full-matrix least squares / difference Fourier cycles were performed, which located the remaining non-hydrogen atoms. All nonhydrogen atoms were refined with anisotropic displacement parameters. The hydrogen atoms were placed in ideal positions and refined as riding atoms with relative isotropic displacement parameters. The structure was solved and refined, using the space group Pbca, with Z = 8 for the formula unit, C10H14IN2O2. The final anisotropic full-matrix least-squares refinement on F2 with 136 variables converged at R1 = 1.72%, for the observed data and wR2 = 6.94% for all data. The goodness-of-fit was 1.301. The largest peak in the final difference electron density synthesis was 0.694 e-/Å 3 and the largest hole was-0.859 e-/Å 3 with an RMS deviation of 0.231 e-/Å 3. On the basis of the final model, the calculated density was 1.943 g/cm 3 and F(000), 1256 e-. The remaining electron density is minuscule and located on bonds. Additional crystal and refinement information is summarized in Table S1. S6 Table S1. Crystal Data and Structure Refinement for Nitroxide 1 (label: s16116). Empirical formula C10 H14 I N2 O2 Formula weight 321.13 Crystal color, shape, size yellow block, 0.24 × 0.23 × 0.12 mm 3 Temperature 173(2) K Wavelength 0.71073 Å Crystal system, space group Orthorhombic, Pbca Unit cell dimensions a = 8.3415(4) Å = 90°. b = 10.1215(4) Å = 90°.