Nanopure system

All chemicals were purchased from commercial sources, including DMSO (purity, ≥99.9%; EMD Chemical), and deionized water was used (specific conductance, 18.2 MΩ cm; Nanopure system, Siemens). The EAL protein from S. typhimurium overexpressed in Escherichia coli overexpression system and purified as described,^{34 (link)–35 (link)} with modifications.^{33 (link)} The specific activity of purified EAL with aminoethanol as substrate was 20 mmol/min/mg (T=298 K, P=1 atm), as determined by using the coupled assay with alcohol dehydrogenase and NADH.^{36 (link)} Protein samples included 10 mM potassium phosphate buffer (pH 7.5), 2–20 μM EAL protein (20 μM was the standard concentration), and 0.2 mM TEMPOL spin probe (4-hydroxy-TEMPO, Sigma-Aldrich) in a final volume of 0.3 ml. When present, DMSO was added to 1% v/v in the final volume of 0.3 ml. Protein and 0% DMSO solution samples were prepared aerobically, on ice in small vials, mixed, and loaded into 4 mm outer diameter EPR tubes (Wilmad-LabGlass). The samples were frozen by immersion in T=140 K isopentane solution. This method has a characteristic cooling rate of 10 K/s.^{33 (link)} Samples were transferred to liquid nitrogen for storage. Solution (no protein) samples containing 1% v/v DMSO were placed in 2mm outer diameter EPR tubes, because of their lossiness at T>210 K.

All chemicals were purchased from commercial sources, including DMSO (purity, ≥99.9%; EMD Chemical), and deionized water was used (resistivity, 18.2 MΩ·cm; Nanopure system, Siemens). The EAL protein from S. typhimurium was obtained from an Escherichia coli overexpression systems and purified as described,^{30 (link)–31 (link)} with modifications.^{29 (link)} The specific activity of purified EAL with aminoethanol as substrate was 20 μmol/min/mg (T=298 K, P=1 atm), as determined by using the coupled assay with alcohol dehydrogenase and NADH.^{32 (link)} Protein samples included 10 mM potassium phosphate buffer (pH 7.5), 20 μM EAL protein, and 0.2 mM TEMPOL spin probe (4-hydroxy-TEMPO, Sigma-Aldrich; added from a freshly-prepared stock solution in water) in a final volume of 0.3 ml. When present, DMSO was added to 0.5, 2.0, and 4.0% v/v, respectively, relative to the final, 0.3 ml volume of the EPR sample. The EPR samples were prepared aerobically, on ice in small vials, mixed, and loaded into 4 mm outer diameter EPR tubes (Wilmad-LabGlass). The samples were frozen by immersion in isopentane solution at T=140 K. This method has a characteristic cooling rate of 10 K/s.^{29 (link)} Samples were transferred to liquid nitrogen for storage. Samples without EAL protein were prepared by the same methods, in 2 mm outer diameter EPR tubes.

All chemicals were purchased from commercial sources, including DMSO (purity, ≥99.9%; EMD Chemical), 4-maleimido-TEMPO (4MT, Sigma-Aldrich). Deionized water was used to prepare aqueous solutions (specific conductance, 18.2 ΜΩ cm; Nanopure system, Siemens). The wt EAL protein was overexpressed in Escherichia coli and purified as described,[7 (link), 33 (link)] with modifications.[12 (link)] Enzyme activity of purified EAL with aminoethanol as substrate was determined at T=298 K and P = 1 atm by using the coupled assay with alcohol dehydrogenase and NADH.[34 ]