Electron spin echo decay as a probe of aminoxyl environment in spin-labeled mutants of human carbonic anhydrase II †

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Abstract
Genetically-engineered human carbonic anhydrase II mutants have been prepared with cysteine introduced at selected locations and spin-labeled with an aminoxyl (formerly known as nitroxide) radical. Two-pulse electron spin echo data have been obtained for samples in 1∶1 water–glycerol employing a Bruker ESP380E spectrometer. Data obtained at 11 and 40 K are fitted to the function Y(τ) = Y(0)· exp[–(2τ/Tm)x]. Tm = 4.4 to 4.1 µs with x > 2 for labels near the surface, but the decay shape changes to Tm = 2 µs, x = 1 for a label buried in a hydrophobic region of the protein. To identify characteristics of the spin label environment that impact Tm and x, 0.1 to 0.5 mM solutions of aminoxyls are examined in a series of glassy solvents. At these spin label concentrations spin echo dephasing is dominated by interaction with solvent protons. For solvents that do not contain methyl groups 1/Tm increases as solvent proton concentration increases. The smallest values of x and of Tm are observed for solvents with the least sterically hindered methyl groups. In samples of spin-labeled engineered proteins the aminoxyl-probe is generally used to explore local motions near room temperature. The data presented here indicate that the shape of the echo decay obtained at low temperature is a sensitive indicator of the proton environment of the spin-label. The combination of lineshape studies at room temperature and spin echo studies at low temperature provide complementary information in spin labeling studies of protein folding and protein–protein interaction.

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