Electron spin echo decay as a probe of aminoxyl environment in spin-labeled mutants of human carbonic anhydrase II
Electron spin echo decay as a probe of aminoxyl environment in spin-labeled mutants of human carbonic anhydrase II
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DOI：
10.1039/a702470c
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发表时间：
1997-12-01
期刊：
影响因子：
--
通讯作者：
Carlsson, U
中科院分区：
文献类型：
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作者：
Lindgren, M;Eaton, GR;Carlsson, U
基因工程的人碳酸氢酶II突变体是用半胱氨酸在选定的位置引入，并用氨氧基(以前称为氮氧化物)自旋标记的。用Bruker ESP380E谱仪获得了1：1水-甘油溶液的双脉冲电子自旋回波数据。在11K和40K下得到的数据符合函数Y(Tau)=Y(0)。EXP[-(2tau/T-m)(X)]。对于接近表面的标记物，T-m=4.4~4.1亩S，具有x>；2，但对于埋在蛋白质疏水区的标记物，衰变形状变为T-m=2亩S，x=1。为了确定影响T-m和x的自旋标签环境的特征，在一系列玻璃溶剂中检测了0.1到0.5 mm的氨基氧基溶液。在这些自旋标记浓度下，自旋回波退相主要受与溶剂质子的相互作用的影响。对于不含甲基的溶剂，1/T-m随溶剂质子浓度的增加而增大。对于空间位阻最小的甲基的溶剂，x和T-m的值最小。在自旋标记的工程蛋白样品中，氨基氧基探针通常用于探索室温附近的局部运动。本文给出的数据表明，在低温下获得的回波衰变的形状是自旋标签质子环境的灵敏指示器。室温下的线型研究和低温下的自旋回波研究相结合，在蛋白质折叠和蛋白质相互作用的自旋标记研究中提供了补充信息。
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(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 decay shape changes to T-m = 2 mu s, x = 1 for a label buried in a hydrophobic 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 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/T-m increases as solvent proton concentration increases. The smallest values of x and of T-m 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.