ELECTRON-SPIN ECHO DECAY AS A PROBE OF AMINOXYL ENVIRONMENT IN SPIN-LABELED MUTANTS OF HUMAN CARBONIC-ANHYDRASE-II

Genetically-engineered human carbonic anhydrase II mutants have been p repared with cysteine introduced at selected locations and spin-labele d with an aminoxyl (formerly known as nitroxide) radical. Two-pulse el ectron spin echo data have been obtained for samples in 1:1 water-glyc erol employing a Bruker ESP380E spectrometer. Data obtained at 11 and 40 K are fitted to the function Y(tau) = Y(0). exp[-(2 tau/T-m)(x)]. T -m = 4.4 to 4.1 mu s with x > 2 for labels near the surface, but the d ecay shape changes to T-m = 2 mu s, x = 1 for a label buried in a hydr ophobic region of the protein. To identify characteristics of the spin label environment that impact T-m and x, 0.1 to 0.5 mM solutions of a minoxyls are examined in a series of glassy solvents. At these spin la bel concentrations spin echo dephasing is dominated by interaction wit h solvent protons. For solvents that do not contain methyl groups 1/T- m increases as solvent proton concentration increases. The smallest va lues of x and of T-m are observed for solvents with the least sterical ly hindered methyl groups. In samples of spin-labeled engineered prote ins 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 lineshap e 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.