Cationic Spin Probe Reporting on Thermal Denaturation and Complexation–Decomplexation of BSA with SDS. Potential Applications in Protein Purification Processes

In this work, we present evidence on the suitability of spin probes to report on the thermal treatment of bovine serum albumin (BSA), in the temperature range 293–343 K, and indirectly monitor the release of sodium dodecyl sulfate (SDS) from its complex with BSA using a covalent gel with β-cyclodextrin (β-CD) in the network. The spin probes used, 5- and 7-doxyl-stearic acids (5-DSA, 7-DSA) or 4-(<i>N</i>,<i>N</i>′-dimethyl-<i>N</i>-hexadecyl)­ammonium-2,2′,6,6′-tetramethylpiperidine-1-oxyl iodide (CAT16), present similar, fatty acid-like structural features. Their continuous wave electron paramagnetic resonance (CW-EPR) spectra, however, reflect different dynamics when complexed with BSA: a restricted motion for 5-DSA, almost nonsensitive to the heating/cooling cycle, and a faster temperature-dependent dynamic motion for CAT16. Molecular docking allows us to rationalize these results by revealing the different binding modes of 5-DSA and CAT16. The EPR data on the temperature effect on BSA are supported by circular dichroism results projecting recovery, upon cooling, of the initial binding ability of BSA for samples heated to 323 K. The interactions occurring in BSA/SDS/β-CD systems are investigated by CW-EPR and FT-ESEEM spectroscopies. It is found that the covalent gel containing β-CD can efficiently remove SDS from the BSA/SDS complex. The gel is not permeable to BSA but it can encapsulate SDS, thus yielding the free protein in solution and allowing recovery of the native protein conformation. Collectively, the accrued knowledge supports potential applications in protein purification biotechnological processes

Electron Transfer Oxidation of Benzene and Aerobic Oxidation to Phenol

The activation of very strong C–H bonds, such as those found in benzene, is important also in the quest for new routes for its functionalization. Using the H₅PV₂Mo₁₀O₄₀ polyoxometalate as an electron transfer oxidant in >50% aqueous H₂SO₄ as solvent, the formation of a benzene radicaloid species at RT as probed by visible spectroscopy and by EPR spectroscopy recorded at X-band and W-band, including ELDOR-detected NMR, was verified. The viability of the ET oxidation of benzene is supported by DFT calculations, showing the reaction to be exergonic under these conditions. Furthermore, we show that in the presence of O₂, very selective hydroxylation to phenol took place

Selective Distance Measurements Using Triple Spin Labeling with Gd³⁺, Mn²⁺, and a Nitroxide

Distance measurements by pulse electron paramagnetic resonance techniques, such as double electron–electron resonance (DEER, also called PELDOR), have become an established tool to explore structural properties of biomacromolecules and their assemblies. In such measurements a pair of spin labels provides a single distance constraint. Here we show that by employing three different types of spin labels that differ in their spectroscopic and spin dynamics properties it is possible to extract three independent distances from a single sample. We demonstrate this using the <i>Antennapedia</i> homeodomain orthogonally labeled with Gd³⁺ and Mn²⁺ tags in complex with its cognate DNA binding site labeled with a nitroxide

Small Gd(III) Tags for Gd(III)–Gd(III) Distance Measurements in Proteins by EPR Spectroscopy

The <b>C7-Gd</b> and <b>C8-Gd</b> tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the <b>C7-Gd</b> (<i>S</i> configuration) and <b>C8-Gd</b> (<i>R</i> configuration) tags loaded with Gd­(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd­(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band ¹H-ENDOR (electron–electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron–electron resonance) measurements were insensitive to the <i>R</i> or <i>S</i> configuration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd­(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, the <b>C7-Gd</b> and <b>C8-Gd</b> tags are attractive Gd­(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy